Car based immunotherapy

ABSTRACT

The disclosure provides chimeric antigen receptors (CARs), T cells comprising such CARs, nucleic acids that encode such CARS, and methods of use thereof, e.g., to treat cancer such as B cell malignancies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.provisional application No. 62/002,603 filed May 23, 2014, the contentsof which are incorporated by reference herein in their entirety.

BACKGROUND

The chimeric antigen receptor (CAR) provides a promising approach foradoptive T-cell immunotherapy for cancer. Commonly, CARs comprise asingle chain fragment variable (scFv) region of an antibody or a bindingdomain specific for a tumor associated antigen (TAA) coupled via hingeand transmembrane regions to cytoplasmic domains of T-cell signalingmolecules. The most common lymphocyte activation moieties include aT-cell costimulatory domain in tandem with a T-cell effector functiontriggering (e.g. CD3ζ) moiety. The CAR-mediated adoptive immunotherapyallows CAR-grafted T cells to directly recognize the TAAs on targettumor cells in a non-HLA-restricted manner.

The large majority of patients having B-cell malignancies, including Bcell acute lymphocytic leukemia (B-ALL) and chronic lymphocytic leukemia(CLL), will die from their disease. One approach to treating thesepatients is to genetically modify T cells to target antigens expressedon tumor cells through the expression of CARs. CARs are antigenreceptors that are designed to recognize cell surface antigens in ahuman leukocyte antigen (HLA)-independent manner. Attempts in usinggenetically modified cells expressing CARs to treat these types ofpatients have met with promising success.

In most cancers, tumor-specific antigens are not yet well defined, butin B cell malignancies, CD19 is an attractive tumor target. Expressionof CD19 is restricted to normal and malignant B cells (Uckun, et al.Blood, 1988, 71: 13-29), so that CD19 is a widely accepted target tosafely test CARs,

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIG. 1 provides a schematic of a CAR-4S lentiviral design.

FIG. 2 is a series of photographs that show CAR T cells targetGFP+/B-ALL cells. The top row shows brightfield images. The bottom rowshows GFP images.

FIG. 3 is a series of graphs depicting a CAR T (CFSE) ProliferationAssay.

FIG. 4 is a series of photographs showing confocal microscopy imaging ofCAR T cells targeting CD19+ cancer cells. The arrow points to a CD19leukemic cell that is intact at 0 minutes and apoptotic at 8 minutes.The cell adjacent to and to the left of the leukemic cell is ananti-CD19 CAR T cell.

FIG. 5 is a diagram showing a synthetic inducer of CAR dimerization andapoptosis.

FIG. 6 is a graph showing rapid induction of CAR T apoptosis.

FIG. 7 is a diagram showing Lenti-4S-CAR T cell therapy.

FIG. 8 is a series of graphs of FACS data showing efficient lentiviral(LV) gene transfer into human T cells.

FIG. 9 is a series of graphs of FACS data showing day 5 LV CAR T cells.

FIG. 10 is a graph showing plasma IgG post CAR-T infusion.

FIG. 11 is a graph showing plasma IgA/IgM post CAR-T infusion.

FIG. 12 is a series of graphs showing concentration of IL-6,concentration of IFN-γ, CAR copy number in PBMC, and CAR copy number inBM.

FIG. 13 is a diagram of the treatment protocol used for case study 2.

FIG. 14 is a series of graphs showing serum Uric Acid and Creatininelevels at different time points after infusion.

FIG. 15 is a series of graphs showing serum IL-6 and IFN-γconcentrations at different time points after infusion.

FIG. 16 is a graph showing Ig (IgG, IgA, or IgM) levels at differenttime points after infusion.

FIG. 17 is a series of photographs showing clinical response, before CART cell treatment, 20 days after cell infusion, and 56 days after cellinfusion.

DETAILED DESCRIPTION

The invention relates in part to compositions and methods for treatingcancer including, but not limited to, blood types of cancer. The presentinvention relates to a strategy of adoptive cell transfer of T cellstransduced to express a chimeric antigen receptor (CAR). CARs aremolecules that combine antibody-based specificity for a desired antigen(e.g., tumor antigen) with a T cell receptor-activating intracellulardomain to generate a chimeric protein that exhibits a specificanti-tumor cellular immune activity.

The present invention, in some embodiments, provides for a compositionswhere a CAR, or portions thereof, is fully human, thereby minimizing therisk for a host immune response.

The present invention, in some embodiments, relates generally to the useof T cells genetically modified to stably express a desired CAR. T cellsexpressing a CAR are referred to herein as CAR T cells or CAR modified Tcells. Preferably, the cell can be genetically modified to stablyexpress an antibody binding domain on its surface, conferring novelantigen specificity that is MHC independent. In some instances, the Tcell is genetically modified to stably express a CAR that combines anantigen recognition domain of a specific antibody with an intracellulardomain of the CD3-zeta chain or FcR protein into a single chimericprotein. In a specific example the CAR an antigen binding domain, atransmembrane domain, a costimulatory signaling region, and a CD3 zetasignaling domain, wherein the costimulatory signaling region comprises aCD28 signaling domain, a CD137 signaling domain and a CD27 signalingdomain, along with a self-destructive gene design such as inducedcaspase 9 (iCasp9) gene. In an even more specific embodiment, thearrangement of the elements of the CAR is as follows:

scFv-28-137-27-CD3z-2A-iCasp9The CD28 element may, but not necessarily, comprise at least a portionof the transmembrane domain of the CAR embodiment above. The CAR mayalso include several hinge elements and/or spacer sequences (such asbetween individual domain elements).

Embodiments of the present invention relate to a CAR that incorporates anovel series of domains that provide different functional aspects thatsynergistically work together to improve efficacy. For example, the CARincludes, in some embodiments, an antigen recognition domain, an antigenco-signaling domain that stimulates activity, a survival domain thatincreases T-cell survival, T-cell memory domain, and an effectoractivating domain. Alternatively, the CAR may further include a domainthat induces safety. FIG. 1 provides an example of a series of domainsthat achieve this multi-functionality.

In some embodiments, the CAR of the invention comprises an extracellulardomain having an antigen recognition domain, a transmembrane domain, anda multi-functional cytoplasmic domain. In some embodiments, two CARproteins dimerize (e.g., form homo- or heterodimers) in vivo. In someembodiments, the CAR can comprise a fully human antibody or antibodyfragment, e.g., a scFv. In one embodiment, the transmembrane domain thatnaturally is associated with one of the domains in the CAR is used. Insome embodiments, the transmembrane domain can be selected or modifiedby amino acid substitution to avoid binding of such domains to thetransmembrane domains of the same or different surface membrane proteinsto minimize interactions with other members of the receptor complex. Insome embodiments, the transmembrane domain is a CD8a transmembranedomain.

In some embodiments, the CAR T cells of the invention can be generatedby introducing a lentiviral vector comprising a nucleic acid encoding adesired CAR targeting a tumor antigen into the cells. For example, thelentiviral vector comprises a nucleic acid encoding CAR comprising atumor antigen binding domain (e.g., targets CD19), optionally a CD8ahinge, a transmembrane domain, and CD28, CD137, CD27 and CD3-zetasignaling domains, into the cells. In some embodiments, the CAR T cellsof the invention are able to replicate in vivo resulting in long-termpersistence that can lead to sustained tumor control. In someembodiments, the CAR T cells of the invention can be generated bytransfecting an RNA encoding the desired CAR, into the cells. In someembodiments, the CAR is transiently expressed in the geneticallymodified CAR T cells.

In some embodiments, the invention relates to a CAR comprising a humanantibody, or fragments thereof. The invention is based, in part, uponthe discovery that a CAR comprising an antigen recognition domaincomprising a fully human antibody fragment specifically recognizes tumorantigens. Therefore, in some embodiments, human CARs of the inventioncan be used to treat cancers and other disorders and avoid the risk ofinducing an immune response.

In some embodiments, the invention relates to administering agenetically modified T cell expressing a CAR for the treatment of apatient having cancer or at risk of having cancer using lymphocyteinfusion. Preferably, autologous lymphocyte infusion is used in thetreatment. Autologous PBMCs are collected from a patient in need oftreatment and T cells are activated and expanded using the methodsdescribed herein and known in the art and then infused back into thepatient.

The invention may include using T cells expressing a CAR comprising anantigen binding domain, a transmembrane domain, a costimulatorysignaling region, and a CD3 zeta signaling domain, wherein thecostimulatory signaling region comprises a CD28 signaling domain, aCD137 signaling domain and a CD27 signaling domain. In some embodiments,CAR T cells of the invention can undergo robust in vivo T cell expansionand can establish specific memory cells that persist at high levels foran extended amount of time in blood and bone marrow. In some instances,the CAR T cells of the invention infused into a patient can eliminatecancerous cells in vivo in patients with cancer.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of +20% or +10%, more preferably +5%, even more preferably+1%, and still more preferably +0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

As used herein, the terms “FRa binding domain” may refer to any FRaspecific binding domain, known to one of skilled in the art. In oneexample, FRa binding domain comprises a single-chain variable fragment(scFv) comprising the variable regions of the heavy (V_(H)) and lightchains (V_(L)) of an antibody binding specifically to FRa. Anti-FRaantibodies, antibody fragments, and their variants are well known in theart and fully described in U.S. Patent Publications U.S. 20100055034;U.S. 20090324594; U.S. 20090274697; U.S. 20080260812; U.S. 20060239910;U.S. 20050232919; U.S. 20040235108, all of which are incorporated byreference herein in their entirety. In one embodiment, the FRa bindingdomain is a homologue, a variant, an isomer, or a functional fragment ofan anti-FRa antibody. Each possibility represents a separate embodimentof the present invention.

In some embodiments, a “protein domain” or “domain” refers to a distinctglobular unit that can be identified as such by a structuredetermination method such as X-ray crystallography or NMR, by otherbiophysical methods such as scanning calorimetry according to which aprotein domain melts as a distinct unit (see for example Pabo et al.Proc Natl Acad Sci USA. (1979) 76(4):1608-12), or by sequence similarityto protein domains whose structure has been determined. The SCOPdatabase (Murzin et al. J Mol Biol. (1995) 247(4):536-40) provides theidentification of protein domains so that the domain organization of anew protein can be identified by sequence comparison. Protein domainsmay comprise an amino acid sequence that is sufficient to drive foldingof such a polypeptide into a discrete structure, in which essentiallyall of the rotatable bonds along the main chain of the polypeptide areconstrained to within about 10 degrees.

“Activation”, as used herein, refers to the state of a T cell that hasbeen sufficiently stimulated to induce detectable cellularproliferation. Activation can also be associated with induced cytokineproduction, and detectable effector functions. The term “activated Tcells” refers to, among other things, T cells that are undergoing celldivision.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which specifically binds with an antigen. Antibodies can beintact immunoglobulins derived from natural sources or from recombinantsources and can be immunoreactive portions of intact immunoglobulins.Antibodies are typically tetramers of immunoglobulin molecules. Theantibodies in the present invention may exist in a variety of formsincluding, for example, polyclonal antibodies, monoclonal antibodies,Fv, Fab and F(ab)₂, as well as single chain antibodies, humanantibodies, and humanized antibodies (Harlow et al., 1999, In: UsingAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, ColdSpring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; Bird et al., 1988, Science 242:423-426).

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limitedto, Fab, Fab′, F(ab′)₂, and Fv fragments, linear antibodies, singlechain antibodies (e.g., scFv antibodies), and multispecific antibodiesformed from antibody fragments.

An “antibody heavy chain,” as used herein, refers to the larger of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations.

An “antibody light chain,” as used herein, refers to the smaller of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations, a and X light chains refer tothe two major antibody light chain isotypes.

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

The term “antigen” or “Ag” as used herein is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan will understand thatany DNA, which comprises a nucleotide sequences or a partial nucleotidesequence encoding a protein that elicits an immune response thereforeencodes an “antigen” as that term is used herein. Furthermore, oneskilled in the art will understand that an antigen need not be encodedsolely by a full length nucleotide sequence of a gene. It is readilyapparent that the present invention includes, but is not limited to, theuse of partial nucleotide sequences of more than one gene and that thesenucleotide sequences are arranged in various combinations to elicit thedesired immune response. Moreover, a skilled artisan will understandthat an antigen need not be encoded by a “gene” at all. It is readilyapparent that an antigen can be generated synthesized or can be derivedfrom a biological sample. Such a biological sample can include, but isnot limited to a tissue sample, a tumor sample, a cell or a biologicalfluid.

The term “tumor antigen” as used herein refers to an antigen associatedwith a cancer cell. Examples of tumor antigens include but are notlimited to CD19, CD20, CD22, ROR1, mesothelin, CD33/IL3Ra, c-Met, PSMA,Glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, and MAGE A3 TCR.

The term “anti-tumor effect” as used herein, refers to a biologicaleffect which can be manifested by a decrease in tumor volume, a decreasein the number of tumor cells, a decrease in the number of metastases, anincrease in life expectancy, or amelioration of various physiologicalsymptoms associated with the cancerous condition. An “anti-tumor effect”can also be manifested by the ability of the peptides, polynucleotides,cells and antibodies of the invention in prevention of the occurrence oftumor in the first place.

The term “auto-antigen” means, in accordance with the present invention,any self-antigen which is mistakenly recognized by the immune system asbeing foreign. Auto-antigens comprise, but are not limited to, cellularproteins, phosphoproteins, cellular surface proteins, cellular lipids,nucleic acids, glycoproteins, including cell surface receptors.

The term “autoimmune disease” as used herein is defined as a disorderthat results from an autoimmune response. An autoimmune disease is theresult of an inappropriate and excessive response to a self-antigen.Examples of autoimmune diseases include but are not limited to,Addision's disease, alopecia greata, ankylosing spondylitis, autoimmunehepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type I),dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis,Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolyticanemia, systemic lupus erythematosus, multiple sclerosis, myastheniagravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoidarthritis, sarcoidosis, scleroderma, Sjogren's syndrome,spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema,pernicious anemia, ulcerative colitis, among others.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the individual.

“Allogeneic” refers to a graft derived from a different animal of thesame species. “Xenogeneic” refers to a graft derived from an animal of adifferent species.

The term “cancer” as used herein is defined as a disease characterizedby the rapid and uncontrolled growth of aberrant cells. Cancer cells canspread locally or through the bloodstream and lymphatic system to otherparts of the body. Examples of various cancers include but are notlimited to, breast cancer, prostate cancer, ovarian cancer, cervicalcancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer,liver cancer, brain cancer, lymphoma, leukemia, lung cancer and thelike. In a specific embodiment, cancer refers to B-cell relatedmalignancies.

“Co-stimulatory ligand,” as the term is used herein, includes a moleculeon an antigen presenting cell (e.g., an aAPC, dendritic cell, B cell,and the like) that specifically binds a cognate co-stimulatory moleculeon a T cell, thereby providing a signal which, in addition to theprimary signal provided by, for instance, binding of a TCR/CD3 complexwith an MHC molecule loaded with peptide, mediates a T cell response,including, but not limited to, proliferation, activation,differentiation, and the like. A co-stimulatory ligand can include, butis not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL,OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesionmolecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM,lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist orantibody that binds Toll ligand receptor and a ligand that specificallybinds with B7-H3. A co-stimulatory ligand also encompasses, inter alia,an antibody that specifically binds with a co-stimulatory moleculepresent on a T cell, such as, but not limited to, CD27, CD28, 4-1BB,OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1(LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specificallybinds with CD83.

A “co-stimulatory molecule” refers to the cognate binding partner on a Tcell that specifically binds with a co-stimulatory ligand, therebymediating a co-stimulatory response by the T cell, such as, but notlimited to, proliferation. Co-stimulatory molecules include, but are notlimited to an MHC class I molecule, BTLA and a Toll ligand receptor. A“co-stimulatory signal”, as used herein, refers to a signal, which incombination with a primary signal, such as TCR/CD3 ligation, leads to Tcell proliferation and/or upregulation or downregulation of keymolecules.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate. In contrast, a “disorder”in an animal is a state of health in which the animal is able tomaintain homeostasis, but in which the animal's state of health is lessfavorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

An “effective amount” as used herein, means an amount which provides atherapeutic or prophylactic benefit.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

“Homologous” refers to the sequence similarity or sequence identitybetween two polypeptides or between two nucleic acid molecules. When aposition in both of the two compared sequences is occupied by the samebase or amino acid monomer subunit, e.g., if a position in each of twoDNA molecules is occupied by adenine, then the molecules are homologousat that position. The percent of homology between two sequences is afunction of the number of matching or homologous positions shared by thetwo sequences divided by the number of positions compared ×100. Forexample, if 6 of 10 of the positions in two sequences are matched orhomologous then the two sequences are 60% homologous. By way of example,the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, acomparison is made when two sequences are aligned to give maximumhomology.

The term “immunoglobulin” or “Ig,” as used herein is defined as a classof proteins, which function as antibodies. Antibodies expressed by Bcells are sometimes referred to as the BCR (B cell receptor) or antigenreceptor. The five members included in this class of proteins are IgA,IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present inbody secretions, such as saliva, tears, breast milk, gastrointestinalsecretions and mucus secretions of the respiratory and genitourinarytracts. IgG is the most common circulating antibody. IgM is the mainimmunoglobulin produced in the primary immune response in most subjects.It is the most efficient immunoglobulin in agglutination, complementfixation, and other antibody responses, and is important in defenseagainst bacteria and viruses. IgD is the immunoglobulin that has noknown antibody function, but may serve as an antigen receptor. IgE isthe immunoglobulin that mediates immediate hypersensitivity by causingrelease of mediators from mast cells and basophils upon exposure toallergen.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the compositions and methods ofthe invention. The instructional material of the kit of the inventionmay, for example, be affixed to a container which contains the nucleicacid, peptide, and/or composition of the invention or be shippedtogether with a container which contains the nucleic acid, peptide,and/or composition. Alternatively, the instructional material may beshipped separately from the container with the intention that theinstructional material and the compound be used cooperatively by therecipient.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

A “lentivirus” as used herein refers to a genus of the Retroviridaefamily.

Lenti viruses are unique among the retroviruses in being able to infectnon-dividing cells; they can deliver a significant amount of geneticinformation into the DNA of the host cell, so they are one of the mostefficient methods of a gene delivery vector. HIV, SIV, and FIV are allexamples of lenti viruses. Vectors derived from lenti viruses offer themeans to achieve significant levels of gene transfer in vivo.

By the term “modulating,” as used herein, is meant mediating adetectable increase or decrease in the level of a response in a subjectcompared with the level of a response in the subject in the absence of atreatment or compound, and/or compared with the level of a response inan otherwise identical but untreated subject. The term encompassesperturbing and/or affecting a native signal or response therebymediating a beneficial therapeutic response in a subject, preferably, ahuman.

“Codon-optimized” means that codons relating to a specific amino acidare optimized for translational efficiency of a gene of interest. Codonoptimization typically involves evaluating the gene or sequence ofinterest and substituting the codon with a more prevalent or commoncodon used for the same amino acid in a specific cell or species.Programs used by those in the art to evaluate codon optimization includethose provided by Integrated DNA Technologies, EnCor Biotechnology,Inc., JCat, OptimumGene™ (GenScript USA, Inc., Piscataway, N.J. 08854),etc. The sequences encoding the CAR embodiments described herein may becodon-optimized, which can increase their translational efficiency.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

The term “operably linked” refers to functional linkage between aregulatory sequence and a heterologous nucleic acid sequence resultingin expression of the latter. For example, a first nucleic acid sequenceis operably linked with a second nucleic acid sequence when the firstnucleic acid sequence is placed in a functional relationship with thesecond nucleic acid sequence. For instance, a promoter is operablylinked to a coding sequence if the promoter affects the transcription orexpression of the coding sequence. Generally, operably linked DNAsequences are contiguous and, where necessary to join two protein codingregions, in the same reading frame.

The term “overexpressed” tumor antigen or “overexpression” of the tumorantigen is intended to indicate an abnormal level of expression of thetumor antigen in a cell from a disease area like a solid tumor within aspecific tissue or organ of the patient relative to the level ofexpression in a normal cell from that tissue or organ. Patients havingsolid tumors or a hematological malignancy characterized byoverexpression of the tumor antigen can be determined by standard assaysknown in the art.

“Parenteral” administration of an immunogenic composition includes, e.g;subcutaneous (s.c), intravenous (i.v.), intramuscular (i.m.), orintrasternal injection, or infusion techniques.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In certain non-limiting embodiments, the patient, subject or individualis a human.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR™, and thelike, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

The term “promoter” as used herein is defined as a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa polynucleotide sequence.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner. A “constitutive” promoteris a nucleotide sequence which, when operably linked with apolynucleotide which encodes or specifies a gene product, causes thegene product to be produced in a cell under most or all physiologicalconditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell substantially only whenan inducer which corresponds to the promoter is present in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide encodes or specified by a gene,causes the gene product to be produced in a cell substantially only ifthe cell is a cell of the tissue type corresponding to the promoter.

By the term “specifically binds,” as used herein with respect to anantibody, is meant an antibody which recognizes a specific antigen, butdoes not substantially recognize or bind other molecules in a sample.For example, an antibody that specifically binds to an antigen from onespecies may also bind to that antigen from one or more species. But,such cross-species reactivity does not itself alter the classificationof an antibody as specific. In another example, an antibody thatspecifically binds to an antigen may also bind to different allelicforms of the antigen. However, such cross reactivity does not itselfalter the classification of an antibody as specific. In some instances,the terms “specific binding” or “specifically binding,” can be used inreference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, to mean that the interaction isdependent upon the presence of a particular structure (e.g., anantigenic determinant or epitope) on the chemical species; for example,an antibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

By the term “stimulation,” is meant a primary response induced bybinding of a stimulatory molecule (e.g., a TCR/CD3 complex) with itscognate ligand thereby mediating a signal transduction event, such as,but not limited to, signal transduction via the TCR/CD3 complex.Stimulation can mediate altered expression of certain molecules, such asdownregulation of TGF-β, and/or reorganization of cytoskeletalstructures, and the like.

A “stimulatory molecule,” as the term is used herein, means a moleculeon a T cell that specifically binds with a cognate stimulatory ligandpresent on an antigen presenting cell.

A “stimulatory ligand,” as used herein, means a ligand that when presenton an antigen presenting cell (e.g., an aAPC, a dendritic cell, aB-cell, and the like) can specifically bind with a cognate bindingpartner (referred to herein as a “stimulatory molecule”) on a T cell,thereby mediating a primary response by the T cell, including, but notlimited to, activation, initiation of an immune response, proliferation,and the like. Stimulatory ligands are well-known in the art andencompass, inter alia, an MHC Class I molecule loaded with a peptide, ananti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonistanti-CD2 antibody.

The term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals). Examples of subjectsinclude humans, dogs, cats, mice, rats, and transgenic species thereof.

As used herein, a “substantially purified” cell is a cell that isessentially free of other cell types. A substantially purified cell alsorefers to a cell which has been separated from other cell types withwhich it is normally associated in its naturally occurring state. Insome instances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to cell that have been separated from the cells with which theyare naturally associated in their natural state. In some embodiments,the cells are cultured in vitro. In other embodiments, the cells are notcultured in vitro.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression, remission,or eradication of a disease state.

The term “therapeutically effective amount” refers to the amount of thesubject compound that will elicit the biological or medical response ofa tissue, system, or subject that is being sought by the researcher,veterinarian, medical doctor or other clinician. The term“therapeutically effective amount” includes that amount of a compoundthat, when administered, is sufficient to prevent development of, oralleviate to some extent, one or more of the signs or symptoms of thedisorder or disease being treated. The therapeutically effective amountwill vary depending on the compound, the disease and its severity andthe age, weight, etc., of the subject to be treated.

To “treat” a disease as the term is used herein, means to reduce thefrequency or severity of at least one sign or symptom of a disease ordisorder experienced by a subject.

The term “transfected” or “transformed” or “transduced” as used hereinrefers to a process by which exogenous nucleic acid is transferred orintroduced into the host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid. The cell includes the primarysubject cell and its progeny.

The phrase “under transcriptional control” or “operatively linked” asused herein means that the promoter is in the correct location andorientation in relation to a polynucleotide to control the initiation oftranscription by RNA polymerase and expression of the polynucleotide.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.

Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,and the like.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

DESCRIPTION

The present invention provides, in some embodiments, compositions andmethods for treating cancer among other diseases. The cancer may be ahematological malignancy, a solid tumor, a primary or a metastasizingtumor. Alternatively, embodiments of the disclosure may be used to treatother diseases, including infectious diseases.

In some embodiments, the invention provides a cell (e.g., T cell)engineered to express a CAR wherein the CAR T cell exhibits an antitumorproperty. In a preferred embodiment, the CAR is a fully human CAR. TheCAR of the invention can be engineered to comprise an extracellulardomain having an antigen binding domain fused to an intracellularsignaling domain of the T cell antigen receptor complex zeta chain(e.g., CD3 zeta). The CAR of the invention when expressed in a T cellis, in some embodiments, able to redirect antigen recognition based onthe antigen binding specificity. An exemplary antigen is CD19 becausethis antigen is expressed on malignant B-cells. However, the inventionis not limited to targeting CD19. Rather, the invention includes anyantigen binding moiety that when bound to its cognate antigen, affects atumor cell so that the tumor cell fails to grow, is prompted to die, orotherwise is affected so that the tumor burden in a patient isdiminished or eliminated. The antigen binding moiety is, in someembodiments, fused with an intracellular domain from multiplecostimulatory molecules and a zeta chain.

In some embodiments, the CAR of the invention comprises an arrangementas follows:

scFv-28-137-27-CD3z-2A-iCasp9

In some embodiments, the above exemplary, non-limiting arrangement isfrom left to right, the N-terminus to C-terminus of the CAR. The CAR maycomprise or further comprise any other combination of elements asdescribed herein.

Composition

The present invention, in some embodiments, provides a chimeric antigenreceptor (CAR) comprising an extracellular and intracellular domain. Insome embodiments, the CAR of the invention is fully human. Theextracellular domain comprises, in some embodiments, a target-specificbinding element otherwise referred to as an antigen binding moiety. Theintracellular domain or otherwise the cytoplasmic domain comprises, acostimulatory signaling region and a zeta chain portion. Thecostimulatory signaling region refers to a portion of the CAR comprisingthe intracellular domain of a costimulatory molecule. Costimulatorymolecules are cell surface molecules other than antigens receptors ortheir ligands that are required for an efficient response of lymphocytesto antigen.

It is to be understood that a CAR can include a region (e.g., an antigenbinding domain, a transmembrane domain, a cytoplasmic domain, asignaling domain, a safety domain and/or a linker, or any combinationthereof) having a sequence provided herein or a variant thereof or afragment of either one thereof (e.g., a variant and/or fragment thatretains the function required for the CAR activity) can be included in aCAR protein as described herein. In some embodiments, a variant has 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes relative to theillustrated sequence. In some embodiments, a variant has a sequence thatis at least 80%, at least 85%, at least 90%, 90%-95%, at least 95% or atleast 99% identical to the illustrated sequence. In some embodiments, afragment is 1-5, 5-10, 10-20, 20-30, 30-40, or 40-50 amino acids shorterthan a sequence provided herein. In some embodiments, a fragment isshorter at the N-terminal, C-terminal, or both terminal regions of thesequence provided. In some embodiments, a fragment contains 80%-85%,85%-90%, 90%-95%, or 95%-99% of the number of amino acids in a sequenceprovided herein.

Between the extracellular domain and the transmembrane domain of theCAR, or between the cytoplasmic domain and the transmembrane domain ofthe CAR, there may be incorporated a spacer domain. As used herein, theterm “spacer domain” generally means any oligo- or polypeptide thatfunctions to link the transmembrane domain to, either the extracellulardomain or, the cytoplasmic domain in the polypeptide chain. A spacerdomain may comprise up to 300 amino acids, preferably 10 to 100 aminoacids and most preferably 25 to 50 amino acids.

In some embodiments, the spacer and/or hinge sequences of the CAR areselected from one or more of the following spacer sequences or hingesequences:

Spacer Sequences: (SEQ ID NO: 1) GGGGS (SEQ ID NO: 2) GGGGSGGGGS(SEQ ID NO: 3) GGGGS x3 GS18: (SEQ ID NO: 4) GSTSGGGSGGGSGGGGSS 218S:(SEQ ID NO: 5) GSTSGSGKPGSSEGSTKG GS8: (SEQ ID NO: 6) GGGGSGGGHinge Sequences: Native: (SEQ ID NO: 7) VEPKSCDKTHTCPPCP C233S:(SEQ ID NO: 8) LDPKSSDKTHTCPPCP C233P: (SEQ ID NO: 9) VEPKSPDKTHTCPPCPDe1ta5: (SEQ ID NO: 10) LDKTHTCPPCP

Antigen Binding Moiety

In some embodiments, the CAR of the invention comprises atarget-specific binding element otherwise referred to as an antigenbinding moiety or antigen binding domain. The choice of moiety dependsupon the type and number of ligands that define the surface of a targetcell. For example, the antigen binding domain may be chosen to recognizea ligand that acts as a cell surface marker on target cells associatedwith a particular disease state. Thus examples of cell surface markersthat may act as ligands for the antigen moiety domain in the CAR of theinvention include those associated with viral, bacterial and parasiticinfections, autoimmune disease and cancer cells. In some embodiments,the CAR of the invention can be engineered to target a tumor antigen ofinterest by way of engineering a desired antigen binding moiety thatspecifically binds to an antigen on a tumor cell. In the context of thepresent invention, “tumor antigen” or “hyperproliferative disorderantigen” or “antigen associated with a hyperproliferative disorder,”refers to antigens that are common to specific hyperproliferativedisorders such as cancer. The antigens discussed herein are merelyincluded by way of example. The list is not intended to be exclusive andfurther examples will be readily apparent to those of skill in the art.

The antigen binding domain of the CAR may target, for example, CD19,CD20, CD22, ROR1, mesothelin, CD33/IL3Ra, c-Met, CD37 PSMA, GlycolipidF77, HER2, EGFRvIII, GD-2, NY-ESO-1 TCR, and MACE A3 TCR. Alternatively,the antigen binding domain portion of the CAR targets an antigen thatincludes but is not limited to FRalpha, CD24, CD44, CD133, CD166, epCAM,CA-125, HE4, Oval, estrogen receptor, progesterone receptor, HER-2/neu,uPA, PAI-1, and the like. Other antigens specific for cancer that may betargeted at taught in PCT publication No. WO2013/123061 (page 20).

The antigen binding domain can be any domain that binds to the antigenincluding but not limited to monoclonal antibodies, single chainantibodies (e.g., scFvs), polyclonal antibodies, synthetic antibodies,human antibodies, humanized antibodies, and fragments thereof. In someinstances, it is beneficial for the antigen binding domain to be derivedfrom the same species in which the CAR will ultimately be used in. Forexample, for use in humans, it may be beneficial for the antigen bindingdomain of the CAR to comprise a human antibody or fragment thereof.Thus, in one embodiment, the antigen biding domain portion comprises ahuman antibody or a fragment thereof.

For in vivo use of antibodies in humans, it may be preferable to usehuman antibodies. Completely human antibodies are particularly desirablefor therapeutic treatment of human subjects. Human antibodies can bemade by a variety of methods known in the art including phage displaymethods using antibody libraries derived from human immunoglobulinsequences, including improvements to these techniques. See, also, U.S.Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO98/50433, WO 98/24893, WO98/16654, WO 96/34096, WO 96/33735, andWO91/10741; each of which is incorporated herein by reference in itsentirety. A human antibody can also be an antibody wherein the heavy andlight chains are encoded by a nucleotide sequence derived from one ormore sources of human DNA. Human antibodies can also be produced usingtransgenic mice which are incapable of expressing functional endogenousimmunoglobulins, but which can express human immunoglobulin genes. Forexample, the human heavy and light chain immunoglobulin gene complexesmay be introduced randomly or by homologous recombination into mouseembryonic stem cells. Alternatively, the human variable region, constantregion, and diversity region may be introduced into mouse embryonic stemcells in addition to the human heavy and light chain genes. The mouseheavy and light chain immunoglobulin genes may be renderednon-functional separately or simultaneously with the introduction ofhuman immunoglobulin loci by homologous recombination. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region (JH) gene in chimeric and germ-line mutant miceresults in complete inhibition of endogenous antibody production. Themodified embryonic stem cells are expanded and microinjected intoblastocysts to produce chimeric mice. The chimeric mice are then bred toproduce homozygous offspring which express human antibodies. Thetransgenic mice are immunized in the normal fashion with a selectedantigen, e.g., all or a portion of a polypeptide of the invention.

Antibodies directed against an antigen can be obtained from theimmunized, transgenic mice using conventional hybridoma technology. Thehuman immunoglobulin transgenes harbored by the transgenic micerearrange during B cell differentiation, and subsequently undergo classswitching and somatic mutation. Thus, using such a technique, it ispossible to produce therapeutically useful IgG, IgA, IgM and IgEantibodies, including, but not limited to, IgG1 (gamma 1) and IgG3. Fora detailed discussion of this technology for producing human antibodiesand human monoclonal antibodies and protocols for producing suchantibodies, see, e.g., PCT Publication Nos. WO2014/055771, WO 98/24893,WO 96/34096, and WO 96/33735; and U.S. Pat. Nos. 5,413,923; 5,625,126;5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and 5,939,598,each of which is incorporated by reference herein in their entirety.

A “humanized” antibody retains a similar antigenic specificity as theoriginal antibody, i.e., in the present invention, the ability to bind,for example, CD19.

In some embodiments, the antigen binding moiety portion of the CAR ofthe invention targets CD19. Preferably, in some embodiments, the antigenbinding moiety portion in the CAR of the invention is a fully humananti-CD19 scFV. Exemplary CD19 scFVs are taught in U.S. Patent NumbersU.S. Pat. Nos. 7,902,338 and 7,109,304, and in U.S. PublishedApplication Numbers US20070178103 and US20130287748, and in PCTPublished Application Number WO2014153270A1, each of which areincorporated herein by reference with respect to the teachings relatedto CD19 scFVs. Exemplary CD19 scFVs are provided in the sequences of SEQID NOs: 3445. In some embodiments, the CD19 scFV comprises the sequenceof any one of SEQ ID NOs: 34-45 or a variant thereof that is at least80%, 85%, 90%, 95%, 98% or 99% identical to any one of SEQ ID NOs:34-45.

Transmembrane Domain

With respect to the transmembrane domain, the CAR can be designed tocomprise a transmembrane domain that is fused to the extracellulardomain of the CAR. In one embodiment, the transmembrane domain thatnaturally is associated with one of the domains in the CAR is used. Insome instances, the transmembrane domain can be selected or modified byamino acid substitution to avoid binding of such domains to thetransmembrane domains of the same or different surface membrane proteinsto minimize interactions with other members of the receptor complex.

The transmembrane domain may be derived either from a natural or from asynthetic source. Where the source is natural, the domain may be derivedfrom any membrane-bound or transmembrane protein. Transmembrane regionsof particular use in this invention may be derived from (e.g., compriseat least the transmembrane region(s) of) the alpha, beta or zeta chainof the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9,CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.In some instances, a variety of human hinges can be employed as wellincluding the human Ig (immunoglobulin) hinge. Transmembrane domains canbe identified using any method known in the art or described herein,e.g., by using the UniProt Database.

In some embodiments, the transmembrane domain may be synthetic, in whichcase it will comprise predominantly hydrophobic residues such as leucineand valine. Preferably a triplet of phenylalanine, tryptophan and valinewill be found at each end of a synthetic transmembrane domain.Optionally, a short oligo- or polypeptide linker, preferably between 2and 10 amino acids in length may form the linkage between thetransmembrane domain and the cytoplasmic signaling domain of the CAR. Aglycine-serine doublet provides a particularly suitable linker.

In some embodiments, the transmembrane domain in the CAR of theinvention is the CD8 transmembrane domain. Sequences of CD8 for thispurposes are taught in PCT pub no. WO2014/055771.

In some embodiments, the transmembrane domain in the CAR of theinvention is the CD28 transmembrane domain.

In some embodiments, the transmembrane domain in the CAR of thedisclosure is a CD28 transmembrane domain. An exemplary sequence of CD28is provided below, as well as an exemplary transmembrane domainsequence. In some embodiments, the CD28 transmembrane domain comprisesthe exemplary transmembrane domain sequence below, or a fragment orvariant thereof that is capable of anchoring a CAR comprising thesequence to a cell membrane. One skilled in the art would appreciatethat the full transmembrane domain, or portion thereof, may beimplemented with the cytoplasmic domain, or a portion thereof. In someembodiments, the transmembrane and cytoplasmic domains used would becontiguous portions of the CD28 sequence.

CD28 (amino acids 19-220) (SEQ ID NO: 11)NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSCD28 (amino acids 153-179, transmembrane domain) (SEQ ID NO: 12)FWVLVVVGGVLACYSLLVTVAFIIFWV

In some embodiments, the CAR of the disclosure is comprises a region ofCD28 that contains all or part of an extracellular domain, all or partof a transmembrane domain and all or part of a cytoplasmic domain. Anexemplary sequence of a region of CD28 for inclusion in a CAR isprovided below. In some embodiments, the CD28 transmembrane domaincomprises the exemplary transmembrane domain sequence below, or afragment or variant thereof that is capable of anchoring a CARcomprising the sequence to a cell membrane.

CD28 region (SEQ ID NO: 13)IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDF AAYRSAS

In some embodiments, the transmembrane domain in the CAR of thedisclosure is a CD27 transmembrane domain. An exemplary sequence of CD27is provided below. One skilled in the art would appreciate that the fulltransmembrane domain of CD27, or portion thereof, may be implementedwith the cytoplasmic domain, or a portion thereof. In some embodiments,the domains would be contiguous portions of the CD27 sequence.

CD27 (amino acids 20-260) (SEQ ID NO: 14)ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMELVFTLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP

In some embodiments, the transmembrane domain of the CAR of thedisclosure comprises a hinge domain such as a CD8 hinge domain. See PCTpub No. WO2014/055771, which teaches an exemplary CD8 hinge domainsequence. An exemplary CD8 hinge domain sequence is also provided below.In some embodiments, the CD8 hinge domain comprises the exemplarysequence below, or a fragment or variant thereof that is capable ofproviding flexibility to or preventing steric hindrance of the CAR orthe domain(s) attached to the hinge domain. In some instances, a varietyof human hinges can be employed as well including the human Ig(immunoglobulin) hinge.

CD8 hinge domain (SEQ ID NO: 15)AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

Cytoplasmic Domain

In some embodiments, the cytoplasmic domain or otherwise theintracellular signaling domain of the CAR of the invention isresponsible for activation of at least one of the normal effectorfunctions of the immune cell in which the CAR has been placed in. Theterm “effector function” refers to a specialized function of a cell.Effector function of a T cell, for example, may be cytolytic activity orhelper activity including the secretion of cytokines. Thus the term“intracellular signaling domain” refers to the portion of a proteinwhich transduces the effector function signal and directs the cell toperform a specialized function. While usually the entire intracellularsignaling domain can be employed, in many cases it is not necessary touse the entire chain. To the extent that a truncated portion of theintracellular signaling domain is used, such truncated portion may beused in place of the intact chain as long as it transduces the effectorfunction signal. The term intracellular signaling domain is thus meantto include any truncated portion of the intracellular signaling domainsufficient to transduce the effector function signal.

Examples of intracellular signaling domains for use in the CAR of theinvention include the cytoplasmic sequences of the T cell receptor (TCR)and co-receptors that act in concert to initiate signal transductionfollowing antigen receptor engagement, as well as any derivative orvariant of these sequences and any synthetic sequence that has the samefunctional capability.

It is known that signals generated through the TCR alone areinsufficient for full activation of the T cell and that a secondary orco-stimulatory signal is also required. Thus, T cell activation can bemediated by two distinct classes of cytoplasmic signaling sequence:those that initiate antigen-dependent primary activation through the TCR(primary cytoplasmic signaling sequences) and those that act in anantigen-independent manner to provide a secondary or co-stimulatorysignal (secondary cytoplasmic signaling sequences). Primary cytoplasmicsignaling sequences regulate primary activation of the TCR complexeither in a stimulatory way, or in an inhibitory way. Primarycytoplasmic signaling sequences that act in a stimulatory manner maycontain signaling motifs which are known as immunoreceptortyrosine-based activation motifs or ITAMs. Examples of IT AM containingprimary cytoplasmic signaling sequences that are of particular use inthe invention include those derived from TCR zeta, FcR gamma, FcR beta,CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.It is particularly preferred that cytoplasmic signaling molecule in theCAR of the invention comprises a cytoplasmic signaling sequence derivedfrom CD3 zeta.

The cytoplasmic domain of the CAR can be designed to comprise theCD3-zeta signaling domain by itself or combined with any other desiredcytoplasmic domain(s) useful in the context of the CAR of the invention.For example, the cytoplasmic domain of the CAR can comprise a CD3 zetachain portion and a costimulatory signaling region. The costimulatorysignaling region refers to a portion of the CAR comprising theintracellular domain of a costimulatory molecule. Thus, while theinvention in exemplified primarily with CD28, CD137 and CD27 as theco-stimulatory signaling element, other additional costimulatoryelements are within the scope of the invention.

The cytoplasmic signaling sequences within the cytoplasmic signalingportion of the CAR of the invention may be linked to each other in arandom or specified order. Optionally, a short oligo- or polypeptidelinker, preferably between 2 and 10 amino acids in length may form thelinkage. A glycine-serine doublet provides a particularly suitablelinker.

In some embodiments, the cytoplasmic domain is designed to comprise thesignaling domain of CD3-zeta and the signaling domain of CD28, CD137 andCD27.

In some embodiments, the cytoplasmic domain comprises a CD27intracellular signaling domain (e.g., CD27 cytoplasmic signalingdomain). In some embodiments, the CD27 signaling domain displayseffector signaling function that enhances immune effector activitiesincluding, but not limited to cell proliferation and cytokineproduction. An exemplary CD27 signaling domain sequence is providedbelow. In some embodiments, the CD27 signaling domain comprises theexemplary sequence below, or a fragment or variant thereof that, whenincluded in a CAR, has the same or an improved function (such ascytolytic activity, cell proliferation or secretion of cytokines)compared to a CAR comprising the exemplary sequence below. The functionmay be tested using any suitable method known in the art.

CD27 signaling domain (SEQ ID NO: 16)QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP

In some embodiments, the cytoplasmic domain comprises a CD137intracellular signaling domain (e.g., CD137 cytoplasmic signalingdomain). An exemplary CD137 signaling domain sequence is provided below.

CD137 signaling domain (SEQ ID NO: 17)KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

In some embodiments, the cytoplasmic domain comprises a CD28intracellular signaling domain (e.g., CD28 cytoplasmic signalingdomain). An exemplary CD28 signaling domain sequence is provided below.

CD28 signaling domain (SEQ ID NO: 18)RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

In some embodiments, the CAR of the disclosure comprises a cytoplasmicsignaling sequence derived from CD3zeta. Examples of CD3 zeta domainsequences are provided below. In some embodiments, the CD3zeta signalingdomain comprises one of the CD3 zeta sequences below, or a fragment orvariant thereof that, when included in a CAR, has the same or animproved function (such as cytolytic activity or secretion of cytokines)compared to a CAR comprising the sequence below.

CD3 zeta signaling domain (SEQ ID NO: 19)RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPRCD3 zeta signaling domain (SEQ ID NO: 20)RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR

The cytoplasmic domain of the CAR can be designed to comprise a CD3-zetasignaling domain combined with any other desired cytoplasmic domain(s)useful in the context of the CAR of the disclosure. For example, thecytoplasmic domain of the CAR can comprise a CD3 zeta domain and acostimulatory signaling region. The costimulatory signaling regionrefers to a portion of the CAR comprising the intracellular domain of acostimulatory molecule. Thus, while the disclosure is exemplifiedprimarily with 4-1BB, CD28, CD137 and CD27 as the co-stimulatorysignaling element, other additional costimulatory elements are withinthe scope of the disclosure. Example sequences of co-stimulatorysignaling regions are shown below.

CD28 (amino acids 180-220, cytoplasmic domain) (SEQ ID NO: 21)RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS4-1BB (CD137) intracellular TRAF binding domain (SEQ ID NO: 22)KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL ICOS intracellular domain(SEQ ID NO: 23) CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLOX40 intracellular domain (SEQ ID NO: 24)ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI CD27 intracellular domain(SEQ ID NO: 25) QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSPCD127 intracellular domain (SEQ ID NO: 26)KRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ

In some embodiments, CAR of the disclosure comprises the apoptosisinducing gene Casp9 or a domain or truncated version thereof. Anexemplary Casp9 sequence and truncated sequence is below. In someembodiments, the CAR comprises a linker (e.g., a peptide linker) betweena CD3 zeta domain and Casp9.

In some embodiments, CAR of the disclosure comprises the apoptosisinducing gene Casp9 or a domain or truncated version thereof. Anexemplary Casp9 sequence and truncated sequence is below. In someembodiments, the CAR comprises a linker (e.g., a peptide linker) betweena CD3 zeta domain and Casp9.

CASP9 amino acid sequence (SEQ ID NO: 27)MDEADRRLLRRCRLRLVEELQVDQLWDALLSRELFRPHMIEDIQRAGSGSRRDQARQLIIDLETRGSQALPLFISCLEDTGQDMLASFLRTNRQAAKLSKPTLENLTPVVLRPEIRKPEVLRPETPRPVDIGSGGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLR KKLFFKTSA truncated CASP9 amino acid sequence (SEQ ID NO: 28)VGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS

In some embodiments, the peptide linker between the CD3 zeta domain andthe Casp9 domain is a 2A peptide linker. A “2A peptide linker” refers toan amino acid sequence encoding the 2A region of a picornavirus (e.g.,foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), Thoseasigna virus (T2A), and porcine teschovirus-1 (P2A)), or a portionthereof. Generally, 2A peptide linkers enable multicistronic proteinexpression by causing ribosome skipping. Examples of 2A linkers areprovided below.

F2A: (SEQ ID NO: 30) VKQTLNFDLLKLAGDVESNPGP E2A: (SEQ ID NO: 31)QCTNYALLKLAGDVESNPGP T2A: (SEQ ID NO: 32) EGRGSLLTCGDVEENPGP P2A:(SEQ ID NO: 33) ATNFSLLKQAGDVEENPGP

In some embodiments, the cytoplasmic domain further comprises an iCasp9domain and/or a FKBP domain. Generally, FK506 binding proteins (FKBP)dimerize in the presence of certain small molecules (e.g., FK1012,FK506, FKCsA, rapamycin, coumermycin, gibberellin, HaXS, etc.). Thus,FKBP are useful for performing chemical dimerization. For example, anFKBP domain may be fused to inactive caspase 9 (iCasp9). In the presenceof certain small molecules, the iCasp9-FKBP protein will dimerize andactivate Casp9, thereby inducing apoptosis of the cell containing theiCasp9-FKBP fusion protein. An exemplary mutated FK506 binding proteinmotif is provided below.

FKBP f36v Amino Acid Sequence

(SEQ ID NO: 29) MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDV ELLKLE

In some embodiments, a CAR comprises or consists of the sequence below,which is broken down by exemplary domains included therein:

Anti-CD19 scFv chain 1 (e.g., a chain 1 of any one of SEQ ID NOs: 34-45)GSTSGSGKPGSSEGSTKG (optional linker; SEQ ID NO: 5)Anti-CD19 scFV chain 2 (e.g., a chain 2 of any one of SEQ ID NOs: 34-45)GSTSGSGKPGSSEGSTKG (optional linker; SEQ ID NO: 5)FWVLVVVGGVLACYSLLVTVAFIIFWV (CD28 transmembrane domain; SEQ ID NO: 12)KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(CD137 cytoplasmic signaling domain; SEQ ID NO: 17) GSTSGSGKPGSSEGSTKG(optional linker; SEQ ID NO: 5)QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP(CD27 cytoplasmic signaling domain; SEQ ID NO: 14)RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR(CD3 zeta signaling domain; SEQ ID NO: 19)2A Linker (e.g., any one of SEQ ID NOs. 30-33)VGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (truncated Casp9, SEQ ID NO: 28).

However, it should be appreciated that other combinations of sequencesdescribed herein can be used in a CAR.

Vectors

In some embodiments, the present invention encompasses a DNA constructcomprising sequences of a CAR, wherein the sequence comprises thenucleic acid sequence of an antigen binding moiety operably linked tothe nucleic acid sequence of an intracellular domain. An exemplaryintracellular domain that can be used in the CAR of the inventionincludes but is not limited to the intracellular domain of CD3-zeta,CD27, CD137, CD28 and the like. In some instances, the CAR can furthercomprise the apoptosis inducing gene Casp9.

The nucleic acid sequences coding for the desired molecules can beobtained using recombinant methods known in the art, such as, forexample by screening libraries from cells expressing the gene, byderiving the gene from a vector known to include the same, or byisolating directly from cells and tissues containing the same, usingstandard techniques. Alternatively, the gene of interest can be producedsynthetically, rather than cloned.

The present invention also provides vectors in which a DNA of thepresent invention is inserted. Vectors derived from retroviruses such asthe lentivirus are suitable tools to achieve long-term gene transfersince they allow long-term, stable integration of a transgene and itspropagation in daughter cells. Lenti viral vectors have the addedadvantage over vectors derived from onco-retroviruses such as murineleukemia viruses in that they can transduce non-proliferating cells,such as hepatocytes. They also have the added advantage of lowimmunogenicity. In another embodiment, the desired CAR can be expressedin the cells by way of transponsons.

In brief summary, the expression of natural or synthetic nucleic acidsencoding CARs is typically achieved by operably linking a nucleic acidencoding the CAR polypeptide or portions thereof to a promoter, andincorporating the construct into an expression vector. The vectors canbe suitable for replication and integration eukaryotes. Typical cloningvectors contain transcription and translation terminators, initiationsequences, and promoters useful for regulation of the expression of thedesired nucleic acid sequence. The expression constructs of the presentinvention may also be used for nucleic acid immunization and genetherapy, using standard gene delivery protocols. Methods for genedelivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346,5,580,859, 5,589,466, incorporated by reference herein in theirentireties. In another embodiment, the invention provides a gene therapyvector.

The nucleic acid can be cloned into a number of types of vectors. Forexample, the nucleic acid can be cloned into a vector including, but notlimited to a plasmid, a phagemid, a phage derivative, an animal virus,and a cosmid. Vectors of particular interest include expression vectors,replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al. (2001, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers, (e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo. A number of retroviralsystems are known in the art. In some embodiments, adenovirus vectorsare used. A number of adenovirus vectors are known in the art. In oneembodiment, lentivirus vectors are used.

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either cooperatively orindependently to activate transcription.

One example of a suitable promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Another example of a suitable promoter is Elongation Growth Factor-1a(EF-1a). However, other constitutive promoter sequences may also beused, including, but not limited to the simian virus 40 (SV40) earlypromoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus(HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avianleukemia virus promoter, an Epstein-Barr virus immediate early promoter,a Rous sarcoma virus promoter, as well as human gene promoters such as,but not limited to, the actin promoter, the myosin promoter, thehemoglobin promoter, and the creatine kinase promoter. Further, theinvention should not be limited to the use of constitutive promoters.Inducible promoters are also contemplated as part of the invention. Theuse of an inducible promoter provides a molecular switch capable ofturning on expression of the polynucleotide sequence which it isoperatively linked when such expression is desired, or turning off theexpression when expression is not desired. Examples of induciblepromoters include, but are not limited to a metallothionine promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter.

In order to assess the expression of a CAR polypeptide or portionsthereof, the expression vector to be introduced into a cell can alsocontain either a selectable marker gene or a reporter gene or both tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors. In other aspects, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers include, for example, antibiotic-resistance genes,such as neo and the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expressionsystems are well known and may be prepared using known techniques orobtained commercially. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al. (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York). A preferred method for the introduction of a polynucleotideinto a host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virus1, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Chloroform is used as the only solventsince it is more readily evaporated than methanol. “Liposome” is ageneric term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,1991 Glycobiology 5: 505-10). However, compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids may assume a micellar structure ormerely exist as nonuniform aggregates of lipid molecules. Alsocontemplated are lipofectamine-nucleic acid complexes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the inhibitor of the presentinvention, in order to confirm the presence of the recombinant DNAsequence in the host cell, a variety of assays may be performed. Suchassays include, for example, “molecular biological” assays well known tothose of skill in the art, such as Southern and Northern blotting,RT-PCR and PCR; “biochemical” assays, such as detecting the presence orabsence of a particular peptide, e.g., by immunological means (ELISAsand Western blots) or by assays described herein to identify agentsfalling within the scope of the invention.

RNA Transfection

In some embodiments, the genetically modified T cells of the inventionare modified through the introduction of RNA. In one embodiment, an invitro transcribed RNA CAR can be introduced to a cell as a form oftransient transfection. The RNA is produced by in vitro transcriptionusing a polymerase chain reaction (PCR)-generated template. DNA ofinterest from any source can be directly converted by PCR into atemplate for in vitro mRNA synthesis using appropriate primers and RNApolymerase. The source of the DNA can be, for example, genomic DNA,plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any otherappropriate source of DNA. The desired template for in vitrotranscription is the CAR of the present invention. For example, thetemplate for the RNA CAR comprises an extracellular domain comprising ananti-CD19 scFv; a transmembrane domain (such as the hinge andtransmembrane domain of CD8a or transmembrane domain of CD28); and acytoplasmic domain comprises the signaling domain of CD3-zeta and thesignaling domain of CD28, CD137 and CD27.

In one embodiment, the DNA to be used for PCR contains an open readingframe. The DNA can be from a naturally occurring DNA sequence from thegenome of an organism. In one embodiment, the DNA is a full length geneof interest of a portion of a gene. The gene can include some or all ofthe 5′ and/or 3′ untranslated regions (UTRs). The gene can include exonsand introns. In one embodiment, the DNA to be used for PCR is a humangene. In another embodiment, the DNA to be used for PCR is a human geneincluding the 5′ and 3′ UTRs. The DNA can alternatively be an artificialDNA sequence that is not normally expressed in a naturally occurringorganism. An exemplary artificial DNA sequence is one that containsportions of genes that are ligated together to form an open readingframe that encodes a fusion protein. The portions of DNA that areligated together can be from a single organism or from more than oneorganism.

Genes that can be used as sources of DNA for PCR include genes thatencode polypeptides that provide a therapeutic or prophylactic effect toan organism or that can be used to diagnose a disease or disorder in anorganism. Preferred genes are genes which are useful for a short termtreatment, or where there are safety concerns regarding dosage or theexpressed gene. For example, for treatment of cancer, autoimmunedisorders, parasitic, viral, bacterial, fungal or other infections, thetransgene(s) to be expressed may encode a polypeptide that functions asa ligand or receptor for cells of the immune system, or can function tostimulate or inhibit the immune system of an organism. In someembodiments, t is not desirable to have prolonged ongoing stimulation ofthe immune system, nor necessary to produce changes which last aftersuccessful treatment, since this may then elicit a new problem. Fortreatment of an autoimmune disorder, it may be desirable to inhibit orsuppress the immune system during a flare-up, but not long term, whichcould result in the patient becoming overly sensitive to an infection.

PCR is used to generate a template for in vitro transcription of mRNAwhich is used for transfection. Methods for performing PCR are wellknown in the art. Primers for use in PCR are designed to have regionsthat are substantially complementary to regions of the DNA to be used asa template for the PCR. “Substantially complementary”, as used herein,refers to sequences of nucleotides where a majority or all of the basesin the primer sequence are complementary, or one or more bases arenon-complementary, or mismatched. Substantially complementary sequencesare able to anneal or hybridize with the intended DNA target underannealing conditions used for PCR. The primers can be designed to besubstantially complementary to any portion of the DNA template. Forexample, the primers can be designed to amplify the portion of a genethat is normally transcribed in cells (the open reading frame),including 5′ and 3′ UTRs. The primers can also be designed to amplify aportion of a gene that encodes a particular domain of interest. In oneembodiment, the primers are designed to amplify the coding region of ahuman cDNA, including all or portions of the 5′ and 3′ UTRs. Primersuseful for PCR are generated by synthetic methods that are well known inthe art.

“Forward primers” are primers that contain a region of nucleotides thatare substantially complementary to nucleotides on the DNA template thatare upstream of the DNA sequence that is to be amplified. “Upstream” isused herein to refer to a location 5, to the DNA sequence to beamplified relative to the coding strand. “Reverse primers” are primersthat contain a region of nucleotides that are substantiallycomplementary to a double-stranded DNA template that are downstream ofthe DNA sequence that is to be amplified. “Downstream” is used herein torefer to a location 3′ to the DNA sequence to be amplified relative tothe coding strand.

Any DNA polymerase useful for PCR can be used in the methods disclosedherein. The reagents and polymerase are commercially available from anumber of sources.

Chemical structures with the ability to promote stability and/ortranslation efficiency may also be used. The RNA preferably has 5′ and3′ UTRs. In one embodiment, the 5′ UTR is between zero and 3000nucleotides in length. The length of 5′ and 3′ UTR sequences to be addedto the coding region can be altered by different methods, including, butnot limited to, designing primers for PCR that anneal to differentregions of the UTRs. Using this approach, one of ordinary skill in theart can modify the 5′ and 3′ UTR lengths required to achieve optimaltranslation efficiency following transfection of the transcribed RNA.

The 5′ and 3′ UTRs can be the naturally occurring, endogenous 5′ and 3′UTRs for the gene of interest. Alternatively, UTR sequences that are notendogenous to the gene of interest can be added by incorporating the UTRsequences into the forward and reverse primers or by any othermodifications of the template. The use of UTR sequences that are notendogenous to the gene of interest can be useful for modifying thestability and/or translation efficiency of the RNA. For example, it isknown that AU-rich elements in 3′ UTR sequences can decrease thestability of mRNA. Therefore, 3′ UTRs can be selected or designed toincrease the stability of the transcribed RNA based on properties ofUTRs that are well known in the art.

In one embodiment, the 5′ UTR can contain the Kozak sequence of theendogenous gene. Alternatively, when a 5′ UTR that is not endogenous tothe gene of interest is being added by PCR as described above, aconsensus Kozak sequence can be redesigned by adding the 5′ UTRsequence. Kozak sequences can increase the efficiency of translation ofsome RNA transcripts, but does not appear to be required for all RNAs toenable efficient translation. The requirement for Kozak sequences formany mRNAs is known in the art. In other embodiments the 5′ UTR can bederived from an RNA virus whose RNA genome is stable in cells. In otherembodiments various nucleotide analogues can be used in the 3′ or 5′ UTRto impede exonuclease degradation of the mRNA.

To enable synthesis of RNA from a DNA template without the need for genecloning, a promoter of transcription should be attached to the DNAtemplate upstream of the sequence to be transcribed. When a sequencethat functions as a promoter for an RNA polymerase is added to the 5′end of the forward primer, the RNA polymerase promoter becomesincorporated into the PCR product upstream of the open reading framethat is to be transcribed. In one preferred embodiment, the promoter isa T7 polymerase promoter, as described elsewhere herein. Other usefulpromoters include, but are not limited to, T3 and SP6 RNA polymerasepromoters. Consensus nucleotide sequences for T7, T3 and SP6 promotersare known in the art.

In a preferred embodiment, the mRNA has both a cap on the 5′ end and a3′ poly(A) tail which determine ribosome binding, initiation oftranslation and stability mRNA in the cell. On a circular DNA template,for instance, plasmid DNA, RNA polymerase produces a long concatamericproduct which is not suitable for expression in eukaryotic cells. Thetranscription of plasmid DNA linearized at the end of the 3′ UTR resultsin normal sized mRNA which is not effective in eukaryotic transfectioneven if it is polyadenylated after transcription.

On a linear DNA template, phage T7 RNA polymerase can extend the 3′ endof the transcript beyond the last base of the template (Schenborn andMierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva andBerzal-Herranz, Eur. J. Biochem., 270: 1485-65 (2003).

The conventional method of integration of polyA/T stretches into a DNAtemplate is molecular cloning. However polyAfT sequence integrated intoplasmid DNA can cause plasmid instability, which is why plasmid DNAtemplates obtained from bacterial cells are often highly contaminatedwith deletions and other aberrations. This makes cloning procedures notonly laborious and time consuming but often not reliable. That is why amethod which allows construction of DNA templates with polyA/T 3′stretch without cloning highly desirable.

The polyA/T segment of the transcriptional DNA template can be producedduring PCR by using a reverse primer containing a polyT tail, such as100T tail (size can be 50-5000 T), or after PCR by any other method,including, but not limited to, DNA ligation or in vitro recombination.Poly(A) tails also provide stability to RNAs and reduce theirdegradation. Generally, the length of a poly(A) tail positivelycorrelates with the stability of the transcribed RNA. In one embodiment,the poly(A) tail is between 100 and 5000 adenosines.

Poly(A) tails of RNAs can be further extended following in vitrotranscription with the use of a poly(A) polymerase, such as E. colipolyA polymerase (E-PAP). In one embodiment, increasing the length of apoly(A) tail from 100 nucleotides to between 300 and 400 nucleotidesresults in about a two-fold increase in the translation efficiency ofthe RNA. Additionally, the attachment of different chemical groups tothe 3′ end can increase mRNA stability. Such attachment can containmodified/artificial nucleotides, aptamers and other compounds. Forexample, ATP analogs can be incorporated into the poly(A) tail usingpoly(A) polymerase. ATP analogs can further increase the stability ofthe RNA.

5′ caps on also provide stability to RNA molecules. In a preferredembodiment, RNAs produced by the methods disclosed herein include a 5′cap. The 5′ cap is provided using techniques known in the art anddescribed herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444(2001); Stepinski, et al., RNA, 7: 1468-95 (2001); Elango, et al.,Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

The RNAs produced by the methods disclosed herein can also contain aninternal ribosome entry site (IRES) sequence. The IRES sequence may beany viral, chromosomal or artificially designed sequence which initiatescap-independent ribosome binding to mRNA and facilitates the initiationof translation. Any solutes suitable for cell electroporation, which cancontain factors facilitating cellular permeability and viability such assugars, peptides, lipids, proteins, antioxidants, and surfactants can beincluded.

RNA can be introduced into target cells using any of a number ofdifferent methods, for instance, commercially available methods whichinclude, but are not limited to, electroporation (Amaxa Nucleofector-II(Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (HarvardInstruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver,Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposomemediated transfection using lipofection, polymer encapsulation, peptidemediated transfection, or biolistic particle delivery systems such as“gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther.,12(8):861-70 (2001).

Genetically Modified T Cells

In some embodiments, the CAR sequences (e.g., nucleic acid sequencesencoding a CAR as described herein) are delivered into cells using aretroviral or lenti viral vector. CAR-expressing retroviral andlentiviral vectors can be delivered into different types of eukaryoticcells as well as into tissues and whole organisms using transduced cellsas carriers or cell-free local or systemic delivery of encapsulated,bound or naked vectors. The method used can be for any purpose wherestable expression is required or sufficient.

In other embodiments, the CAR sequences are delivered into cells usingin vitro transcribed mRNA. In vitro transcribed mRNA CAR can bedelivered into different types of eukaryotic cells as well as intotissues and whole organisms using transfected cells as carriers orcell-free local or systemic delivery of encapsulated, bound or nakedmRNA. The method used can be for any purpose where transient expressionis required or sufficient.

In another embodiment, the desired CAR can be expressed in the cells byway of transponsons.

The disclosed methods can be applied to the modulation of T cellactivity in basic research and therapy, in the fields of cancer, stemcells, acute and chronic infections, and autoimmune diseases, includingthe assessment of the ability of the genetically modified T cell to killa target cancer cell.

The methods also provide the ability to control the level of expressionover a wide range by changing, for example, the promoter or the amountof input RNA, making it possible to individually regulate the expressionlevel. Furthermore, the PCR-based technique of mRNA production greatlyfacilitates the design of the chimeric receptor mRNAs with differentstructures and combination of their domains. For example, varying ofdifferent intracellular effector/costimulator domains on multiplechimeric receptors in the same cell allows determination of thestructure of the receptor combinations which assess the highest level ofcytotoxicity against multi-antigenic targets, and at the same timelowest cytotoxicity toward normal cells.

One advantage of RNA transfection methods of the invention is that RNAtransfection is essentially transient and a vector-free: an RNAtransgene can be delivered to a lymphocyte and expressed thereinfollowing a brief in vitro cell activation, as a minimal expressingcassette without the need for any additional viral sequences. Underthese conditions, integration of the transgene into the host cell genomeis unlikely.

Cloning of cells is not necessary because of the efficiency oftransfection of the RNA and its ability to uniformly modify the entirelymphocyte population. Genetic modification of T cells with invitro-transcribed RNA (IVT-RNA) makes use of two different strategiesboth of which have been successively tested in various animal models.Cells are transfected with in vitro-transcribed RNA by means oflipofection or electroporation. Preferably, it is desirable to stabilizeIVT-RNA using various modifications in order to achieve prolongedexpression of transferred IVT-RNA.

Some IVT vectors are known in the literature which are utilized in astandardized manner as template for in vitro transcription and whichhave been genetically modified in such a way that stabilized RNAtranscripts are produced.

Currently protocols used in the art are based on a plasmid vector withthe following structure: a 5′ RNA polymerase promoter enabling RNAtranscription, followed by a gene of interest which is flanked either 3′and/or 5′ by untranslated regions (UTR), and a 3′ polyadenyl cassettecontaining 50-70 A nucleotides. Prior to in vitro transcription, thecircular plasmid is linearized downstream of the polyadenyl cassette bytype 11 restriction enzymes (recognition sequence corresponds tocleavage site). The polyadenyl cassette thus corresponds to the laterpoly(A) sequence in the transcript. As a result of this procedure, somenucleotides remain as part of the enzyme cleavage site afterlinearization and extend or mask the poly(A) sequence at the 3′ end. Itis not clear, whether this nonphysiological overhang affects the amountof protein produced intracellularly from such a construct.

RNA has several advantages over more traditional plasmid or viralapproaches. Gene expression from an RNA source does not requiretranscription and the protein product is produced rapidly after thetransfection. Further, since the RNA has to only gain access to thecytoplasm, rather than the nucleus, and therefore typical transfectionmethods result in an extremely high rate of transfection. In addition,plasmid based approaches require that the promoter driving theexpression of the gene of interest be active in the cells under study.

In another aspect, the RNA construct can be delivered into the cells byelectroporation. See, e.g., the formulations and methodology ofelectroporation of nucleic acid constructs into mammalian cells astaught in US 2004/0014645, US 2005/0052630A1, US 2005/0070841 A1, US2004/0059285A1, US 2004/0092907A1. The various parameters includingelectric field strength required for electroporation of any known celltype are generally known in the relevant research literature as well asnumerous patents and applications in the field. See e.g., U.S. Pat. Nos.6,678,556, 7,171,264, and 7,173,116. Apparatus for therapeuticapplication of electroporation are available commercially, e.g., theMedPulser™ DNA Electroporation Therapy System (Inovio/Genetronics, SanDiego, Calif.), and are described in patents such as U.S. Pat. Nos.6,567,694; 6,516,223, 5,993,434, 6,181,964, 6,241,701, and 6,233,482;electroporation may also be used for transfection of cells in vitro asdescribed e.g. in US20070128708A1. Electroporation may also be utilizedto deliver nucleic acids into cells in vitro.

Accordingly, electroporation-mediated administration into cells ofnucleic acids including expression constructs utilizing any of the manyavailable devices and electroporation systems known to those of skill inthe art presents an exciting new means for delivering an RNA of interestto a target cell.

Sources of T Cells

Prior to expansion and genetic modification of the T cells of theinvention, a source of T cells may be obtained from a subject. T cellscan be obtained from a number of sources, including peripheral bloodmononuclear cells, bone marrow, lymph node tissue, cord blood, thymustissue, tissue from a site of infection, ascites, pleural effusion,spleen tissue, and tumors. In certain embodiments of the presentinvention, any number of T cell lines available in the art, may be used.In certain embodiments of the present invention, T cells can be obtainedfrom a unit of blood collected from a subject using any number oftechniques known to the skilled artisan, such as Ficoll™ separation. Inone preferred embodiment, cells from the circulating blood of anindividual are obtained by apheresis. The apheresis product typicallycontains lymphocytes, including T cells, monocytes, granulocytes, Bcells, other nucleated white blood cells, red blood cells, andplatelets. In one embodiment, the cells collected by apheresis may bewashed to remove the plasma fraction and to place the cells in anappropriate buffer or media for subsequent processing steps. In oneembodiment of the invention, the cells are washed with phosphatebuffered saline (PBS). In an alternative embodiment, the wash solutionlacks calcium and may lack magnesium or may lack many if not alldivalent cations. Again, surprisingly, initial activation steps in theabsence of calcium lead to magnified activation. As those of ordinaryskill in the art would readily appreciate a washing step may beaccomplished by methods known to those in the art, such as by using asemi-automated “flow-through” centrifuge (for example, the Cobe 2991cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5)according to the manufacturer's instructions. After washing, the cellsmay be resuspended in a variety of biocompatible buffers, such as, forexample, Ca²⁺-free, Mg²⁺-free PBS, PlasmaLyte A, or other salinesolution with or without buffer. Alternatively, the undesirablecomponents of the apheresis sample may be removed and the cells directlyresuspended in culture media.

In another embodiment, T cells are isolated from peripheral bloodlymphocytes by lysing the red blood cells and depleting the monocytes,for example, by centrifugation through a PERCOLL™ gradient or bycounterflow centrifugal elutriation. A specific subpopulation of Tcells, such as CD3⁺, CD28⁺, CD4⁺, CD8⁺, CD45RA⁺, and CD45RO⁺T cells, canbe further isolated by positive or negative selection techniques. Forexample, in one embodiment, T cells are isolated by incubation withanti-CD3/anti-CD28 (i.e., 3×28)-conjugated beads, such as DYNABEADS®M-450 CD3/CD28 T, for a time period sufficient for positive selection ofthe desired T cells. In one embodiment, the time period is about 30minutes. In a further embodiment, the time period ranges from 30 minutesto 36 hours or longer and all integer values there between. In a furtherembodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. Inyet another preferred embodiment, the time period is 10 to 24 hours. Inone preferred embodiment, the incubation time period is 24 hours. Forisolation of T cells from patients with leukemia, use of longerincubation times, such as 24 hours, can increase cell yield. Longerincubation times may be used to isolate T cells in any situation wherethere are few T cells as compared to other cell types, such in isolatingtumor infiltrating lymphocytes (TIL) from tumor tissue or fromimmune-compromised individuals. Further, use of longer incubation timescan increase the efficiency of capture of CD8+ T cells. Thus, by simplyshortening or lengthening the time T cells are allowed to bind to theCD3/CD28 beads and/or by increasing or decreasing the ratio of beads toT cells (as described further herein), subpopulations of T cells can bepreferentially selected for or against at culture initiation or at othertime points during the process. Additionally, by increasing ordecreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on thebeads or other surface, subpopulations of T cells can be preferentiallyselected for or against at culture initiation or at other desired timepoints. The skilled artisan would recognize that multiple rounds ofselection can also be used in the context of this invention. In certainembodiments, it may be desirable to perform the selection procedure anduse the “unselected” cells in the activation and expansion process.“Unselected” cells can also be subjected to further rounds of selection.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. One method is cellsorting and/or selection via negative magnetic immunoadherence or flowcytometry that uses a cocktail of monoclonal antibodies directed to cellsurface markers present on the cells negatively selected. For example,to enrich for CD4⁺ cells by negative selection, a monoclonal antibodycocktail typically includes antibodies to CD14, CD20, CD1 1b, CD16,HLA-DR, and CD8. In certain embodiments, it may be desirable to enrichfor or positively select for regulatory T cells which typically expressCD4⁺, CD25⁺, CD621^(hi), GITR⁺, and FoxP3⁺.

Alternatively, in certain embodiments, T regulatory cells are depletedby anti-C25 conjugated beads or other similar method of selection.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain embodiments, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one embodiment, aconcentration of 2 billion cells/ml is used. In one embodiment, aconcentration of 1 billion cells/ml is used. In a further embodiment,greater than 100 million cells/ml is used. In a further embodiment, aconcentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 millioncells/ml is used. In yet another embodiment, a concentration of cellsfrom 75, 80, 85, 90, 95, or 100 million cells/ml is used. In furtherembodiments, concentrations of 125 or 150 million cells/ml can be used.Using high concentrations can result in increased cell yield, cellactivation, and cell expansion. Further, use of high cell concentrationsallows more efficient capture of cells that may weakly express targetantigens of interest, such as CD28-negative T cells, or from sampleswhere there are many tumor cells present (i.e., leukemic blood, tumortissue, etc.). Such populations of cells may have therapeutic value andwould be desirable to obtain. For example, using high concentration ofcells allows more efficient selection of CD8⁺T cells that normally haveweaker CD28 expression.

In a related embodiment, it may be desirable to use lower concentrationsof cells. By significantly diluting the mixture of T cells and surface(e.g., particles such as beads), interactions between the particles andcells is minimized. This selects for cells that express high amounts ofdesired antigens to be bound to the particles. For example, CD4⁺ T cellsexpress higher levels of CD28 and are more efficiently captured thanCD8⁺ T cells in dilute concentrations. In one embodiment, theconcentration of cells used is 5×10⁶/ml. In other embodiments, theconcentration used can be from about 1×10⁵/ml to 1×10⁶/ml, and anyinteger value in between.

In other embodiments, the cells may be incubated on a rotator forvarying lengths of time at varying speeds at either 2-10° C. or at roomtemperature.

T cells for stimulation can also be frozen after a washing step. Wishingnot to be bound by theory, the freeze and subsequent thaw step providesa more uniform product by removing granulocytes and to some extentmonocytes in the cell population. After the washing step that removesplasma and platelets, the cells may be suspended in a freezing solution.While many freezing solutions and parameters are known in the art andwill be useful in this context, one method involves using PBS containing20% DMSO and 8% human serum albumin, or culture media containing 10%Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitablecell freezing media containing for example, Hespan and PlasmaLyte A, thecells then are frozen to −80° C. at a rate of 1° per minute and storedin the vapor phase of a liquid nitrogen storage tank. Other methods ofcontrolled freezing may be used as well as uncontrolled freezingimmediately at −20° C. or in liquid nitrogen. In certain embodiments,cryopreserved cells are thawed and washed as described herein andallowed to rest for one hour at room temperature prior to activationusing the methods of the present invention.

Also contemplated in the context of the invention is the collection ofblood samples or apheresis product from a subject at a time period priorto when the expanded cells as described herein might be needed. As such,the source of the cells to be expanded can be collected at any timepoint necessary, and desired cells, such as T cells, isolated and frozenfor later use in T cell therapy for any number of diseases or conditionsthat would benefit from T cell therapy, such as those described herein.In one embodiment a blood sample or an apheresis is taken from agenerally healthy subject. In certain embodiments, a blood sample or anapheresis is taken from a generally healthy subject who is at risk ofdeveloping a disease, but who has not yet developed a disease, and thecells of interest are isolated and frozen for later use. In certainembodiments, the T cells may be expanded, frozen, and used at a latertime. In certain embodiments, samples are collected from a patientshortly after diagnosis of a particular disease as described herein butprior to any treatments. In a further embodiment, the cells are isolatedfrom a blood sample or an apheresis from a subject prior to any numberof relevant treatment modalities, including but not limited to treatmentwith agents such as natalizumab, efalizumab, antiviral agents,chemotherapy, radiation, immunosuppressive agents, such as cyclosporin,azathioprine, methotrexate, mycophenolate, and FK506, antibodies, orother immunoablative agents such as CAMPATH, anti-CD3 antibodies,Cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid,steroids, FR901228, and irradiation. These drugs inhibit either thecalcium dependent phosphatase calcineurin (cyclosporine and FK506) orinhibit the p70S6 kinase that is important for growth factor inducedsignaling (rapamycin) (Liu et al., Cell 66:807-815, 1991; Henderson etal., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun.5:763-773, 1993). In a further embodiment, the cells are isolated for apatient and frozen for later use in conjunction with (e.g., before,simultaneously or following) bone marrow or stem cell transplantation, Tcell ablative therapy using either chemotherapy agents such as,fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, orantibodies such as OKT3 or CAMPATH. In another embodiment, the cells areisolated prior to and can be frozen for later use for treatmentfollowing B-cell ablative therapy such as agents that react with CD20,e.g., Rituxan.

In a further embodiment of the present invention, T cells are obtainedfrom a patient directly following treatment. In this regard, it has beenobserved that following certain cancer treatments, in particulartreatments with drugs that damage the immune system, shortly aftertreatment during the period when patients would normally be recoveringfrom the treatment, the quality of T cells obtained may be optimal orimproved for their ability to expand ex vivo. Likewise, following exvivo manipulation using the methods described herein, these cells may bein a preferred state for enhanced engraftment and in vivo expansion.Thus, it is contemplated within the context of the present invention tocollect blood cells, including T cells, dendritic cells, or other cellsof the hematopoietic lineage, during this recovery phase. Further, incertain embodiments, mobilization (for example, mobilization withGM-CSF) and conditioning regimens can be used to create a condition in asubject wherein repopulation, recirculation, regeneration, and/orexpansion of particular cell types is favored, especially during adefined window of time following therapy. Illustrative cell typesinclude T cells, B cells, dendritic cells, and other cells of the immunesystem.

Activation and Expansion of T Cells

Whether prior to or after genetic modification of the T cells to expressa desirable CAR, the T cells can be activated and expanded generallyusing methods as described, for example, in U.S. Pat. Nos. 6,352,694;6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681;7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223;6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application PublicationNo. 20060121005.

Generally, the T cells of the invention are expanded by contact with asurface having attached thereto an agent that stimulates a CD3/TCRcomplex associated signal and a ligand that stimulates a co-stimulatorymolecule on the surface of the T cells. In particular, T cellpopulations may be stimulated as described herein, such as by contactwith an anti-CD3 antibody, or antigen-binding fragment thereof, or ananti-CD2 antibody immobilized on a surface, or by contact with a proteinkinase C activator (e.g., bryostatin) in conjunction with a calciumionophore. For co-stimulation of an accessory molecule on the surface ofthe T cells, a ligand that binds the accessory molecule is used. Forexample, a population of T cells can be contacted with an anti-CD3antibody and an anti-CD28 antibody, under conditions appropriate forstimulating proliferation of the T cells. To stimulate proliferation ofeither CD4⁺ T cells or CD8⁺ T cells, an anti-CD3 antibody and ananti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3,XR-CD28 (Diaclone, Besancon, France) can be used as can other methodscommonly known in the art (Berg et al., Transplant Proc.30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9): 13191328,1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

In certain embodiments, the primary stimulatory signal and theco-stimulatory signal for the T cell may be provided by differentprotocols. For example, the agents providing each signal may be insolution or coupled to a surface. When coupled to a surface, the agentsmay be coupled to the same surface (i.e., in “cis” formation) or toseparate surfaces (i.e., in “trans” formation). Alternatively, one agentmay be coupled to a surface and the other agent in solution. In oneembodiment, the agent providing the co-stimulatory signal is bound to acell surface and the agent providing the primary activation signal is insolution or coupled to a surface. In certain embodiments, both agentscan be in solution. In another embodiment, the agents may be in solubleform, and then cross-linked to a surface, such as a cell expressing Fcreceptors or an antibody or other binding agent which will bind to theagents. In this regard, see for example, U.S. Patent ApplicationPublication Nos. 20040101519 and 20060034810 for artificial antigenpresenting cells (aAPCs) that are contemplated for use in activating andexpanding T cells in the present invention.

In some embodiments, the two agents are immobilized on beads, either onthe same bead, i.e., “cis,” or to separate beads, i.e., “trans.” By wayof example, the agent providing the primary activation signal is ananti-CD3 antibody or an antigen-binding fragment thereof and the agentproviding the co-stimulatory signal is an anti-CD28 antibody orantigen-binding fragment thereof; and both agents are co-immobilized tothe same bead in equivalent molecular amounts. In one embodiment, a 1:1ratio of each antibody bound to the beads for CD4⁺ T cell expansion andT cell growth is used. In certain aspects of the present invention, aratio of anti CD3:CD28 antibodies bound to the beads is used such thatan increase in T cell expansion is observed as compared to the expansionobserved using a ratio of 1:1. In one particular embodiment an increaseof from about 1 to about 3 fold is observed as compared to the expansionobserved using a ratio of 1:1. In one embodiment, the ratio of CD3:CD28antibody bound to the beads ranges from 100:1 to 1:100 and all integervalues there between. In one aspect of the present invention, moreanti-CD28 antibody is bound to the particles than anti-CD3 antibody,i.e., the ratio of CD3:CD28 is less than one. In certain embodiments ofthe invention, the ratio of anti CD28 antibody to anti CD3 antibodybound to the beads is greater than 2:1. In one particular embodiment, a1:100 CD3:CD28 ratio of antibody bound to beads is used. In anotherembodiment, a 1:75 CD3:CD28 ratio of antibody bound to beads is used. Ina further embodiment, a 1:50 CD3:CD28 ratio of antibody bound to beadsis used. In another embodiment, a 1:30 CD3:CD28 ratio of antibody boundto beads is used. In one preferred embodiment, a 1:10 CD3:CD28 ratio ofantibody bound to beads is used. In another embodiment, a 1:3 CD3:CD28ratio of antibody bound to the beads is used. In yet another embodiment,a 3:1 CD3:CD28 ratio of antibody bound to the beads is used.

Ratios of particles to cells from 1:500 to 500:1 and any integer valuesin between may be used to stimulate T cells or other target cells. Asthose of ordinary skill in the art can readily appreciate, the ratio ofparticles to cells may depend on particle size relative to the targetcell. For example, small sized beads could only bind a few cells, whilelarger beads could bind many. In certain embodiments the ratio of cellsto particles ranges from 1:100 to 100:1 and any integer valuesin-between and in further embodiments the ratio comprises 1:9 to 9:1 andany integer values in between, can also be used to stimulate T cells.The ratio of anti-CD3- and anti-CD28-coupled particles to T cells thatresult in T cell stimulation can vary as noted above, however certainpreferred values include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8,1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, and 15:1 with one preferred ratio being at least 1:1particles per T cell. In one embodiment, a ratio of particles to cellsof 1:1 or less is used. In one particular embodiment, a preferredparticle: cell ratio is 1:5. In further embodiments, the ratio ofparticles to cells can be varied depending on the day of stimulation.For example, in one embodiment, the ratio of particles to cells is from1:1 to 10:1 on the first day and additional particles are added to thecells every day or every other day thereafter for up to 10 days, atfinal ratios of from 1:1 to 1:10 (based on cell counts on the day ofaddition). In one particular embodiment, the ratio of particles to cellsis 1:1 on the first day of stimulation and adjusted to 1:5 on the thirdand fifth days of stimulation. In another embodiment, particles areadded on a daily or every other day basis to a final ratio of 1:1 on thefirst day, and 1:5 on the third and fifth days of stimulation. Inanother embodiment, the ratio of particles to cells is 2:1 on the firstday of stimulation and adjusted to 1:10 on the third and fifth days ofstimulation. In another embodiment, particles are added on a daily orevery other day basis to a final ratio of 1:1 on the first day, and 1:10on the third and fifth days of stimulation. One of skill in the art willappreciate that a variety of other ratios may be suitable for use in thepresent invention. In particular, ratios will vary depending on particlesize and on cell size and type.

In further embodiments of the present invention, the cells, such as Tcells, are combined with agent-coated beads, the beads and the cells aresubsequently separated, and then the cells are cultured. In analternative embodiment, prior to culture, the agent-coated beads andcells are not separated but are cultured together. In a furtherembodiment, the beads and cells are first concentrated by application ofa force, such as a magnetic force, resulting in increased ligation ofcell surface markers, thereby inducing cell stimulation.

By way of example, cell surface proteins may be ligated by allowingparamagnetic beads to which anti-CD3 and anti-CD28 are attached (3×28beads) to contact the T cells. In one embodiment the cells (for example,10⁴ to 10⁹ T cells) and beads (for example, DYNABEADS® M-450 CD3/CD28 Tparamagnetic beads at a ratio of 1:1) are combined in a buffer,preferably PBS (without divalent cations such as, calcium andmagnesium). Again, those of ordinary skill in the art can readilyappreciate any cell concentration may be used. For example, the targetcell may be very rare in the sample and comprise only 0.01% of thesample or the entire sample (i.e., 100%) may comprise the target cell ofinterest. Accordingly, any cell number is within the context of thepresent invention. In certain embodiments, it may be desirable tosignificantly decrease the volume in which particles and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and particles. For example, in one embodiment, aconcentration of about 2 billion cells/ml is used. In anotherembodiment, greater than 100 million cells/ml is used. In a furtherembodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45,or 50 million cells/ml is used. In yet another embodiment, aconcentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mlis used. In further embodiments, concentrations of 125 or 150 millioncells/ml can be used. Using high concentrations can result in increasedcell yield, cell activation, and cell expansion. Further, use of highcell concentrations allows more efficient capture of cells that mayweakly express target antigens of interest, such as CD28-negative Tcells. Such populations of cells may have therapeutic value and would bedesirable to obtain in certain embodiments. For example, using highconcentration of cells allows more efficient selection of CD8+ T cellsthat normally have weaker CD28 expression.

In one embodiment of the present invention, the mixture may be culturedfor several hours (about 3 hours) to about 14 days or any hourly integervalue in between. In another embodiment, the mixture may be cultured for21 days. In one embodiment of the invention the beads and the T cellsare cultured together for about eight days. In another embodiment, thebeads and T cells are cultured together for 2-3 days. Several cycles ofstimulation may also be desired such that culture time of T cells can be60 days or more. Conditions appropriate for T cell culture include anappropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or,X-vivo 15, (Lonza)) that may contain factors necessary for proliferationand viability, including serum (e.g., fetal bovine or human serum),interleukin-2 (IL-2), insulin, IFN-7, IL-4, IL-7, GM-CSF, IL-10, IL-12,IL-15, TGFp, and TNF-a or any other additives for the growth of cellsknown to the skilled artisan. Other additives for the growth of cellsinclude, but are not limited to, surfactant, plasmanate, and reducingagents such as N-acetyl-cysteine and 2-mercaptoethanol. Media caninclude RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo20, Optimizer, with added amino acids, sodium pyruvate, and vitamins,either serum-free or supplemented with an appropriate amount of serum(or plasma) or a defined set of hormones, and/or an amount ofcytokine(s) sufficient for the growth and expansion of T cells.Antibiotics, e.g., penicillin and streptomycin, are included only inexperimental cultures, not in cultures of cells that are to be infusedinto a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% C0₂).

T cells that have been exposed to varied stimulation times may exhibitdifferent characteristics. For example, typical blood or apheresedperipheral blood mononuclear cell products have a helper T cellpopulation (¾, CD4⁺) that is greater than the cytotoxic or suppressor Tcell population (T_(c), CD8⁺). Ex vivo expansion of T cells bystimulating CD3 and CD28 receptors produces a population of T cells thatprior to about days 8-9 consists predominately of ¾ cells, while afterabout days 8-9, the population of T cells comprises an increasinglygreater population of Tc cells. Accordingly, depending on the purpose oftreatment, infusing a subject with a T cell population comprisingpredominately of T_(H) cells may be advantageous. Similarly, if anantigen-specific subset of Tc cells has been isolated it may bebeneficial to expand this subset to a greater degree.

Further, in addition to CD4 and CD 8 markers, other phenotypic markersvary significantly, but in large part, reproducibly during the course ofthe cell expansion process. Thus, such reproducibility enables theability to tailor an activated T cell product for specific purposes.

Therapeutic Application

In some embodiments, the present invention encompasses a cell (e.g., Tcell) modified to express a CAR that combines an antigen recognitiondomain of a specific antibody with an intracellular domain of CD3-zeta,CD28, CD27, or any combinations thereof. Therefore, in some instances,the transduced T cell can elicit a CAR-mediated T-cell response.

In some embodiments, the invention provides the use of a CAR to redirectthe specificity of a primary T cell to a tumor antigen. Thus, thepresent invention also provides a method for stimulating a Tcell-mediated immune response to a target cell population or tissue in amammal comprising the step of administering to the mammal a T cell thatexpresses a CAR, wherein the CAR comprises a binding moiety thatspecifically interacts with a predetermined target, a zeta chain portioncomprising for example the intracellular domain of human CD3-zeta, and acostimulatory signaling region. In one embodiment, the present inventionincludes a type of cellular therapy where T cells are geneticallymodified to express a CAR and the CAR T cell is infused to a recipientin need thereof. The infused cell is able to kill tumor cells in therecipient. Unlike antibody therapies, CAR T cells are able to replicatein vivo resulting in long-term persistence that can lead to sustainedtumor control.

In some embodiments, the CAR T cells of the invention can undergo robustin vivo T cell expansion and can persist for an extended amount of time.In another embodiment, the CAR T cells of the invention evolve intospecific memory T cells that can be reactivated to inhibit anyadditional tumor formation or growth. For example, CD19-specific CAR Tcells of the invention can undergo robust in vivo T cell expansion andpersist at high levels for an extended amount of time in blood and bonemarrow and form specific memory T cells. Without wishing to be bound byany particular theory, CAR T cells may differentiate in vivo into acentral memory-like state upon encounter and subsequent elimination oftarget cells expressing the surrogate antigen.

Without wishing to be bound by any particular theory, the anti-tumorimmunity response elicited by the CAR-modified T cells may be an activeor a passive immune response. In addition, the CAR mediated immuneresponse may be part of an adoptive immunotherapy approach in whichCAR-modified T cells induce an immune response specific to the antigenbinding moiety in the CAR. For example, CD19-specific CAR T cells elicitan immune response specific against cells expressing CD19.

While the data disclosed herein specifically disclose lentiviral vectorcomprising human anti-CD19 scFv (e.g. C4 scFv), a transmembrane domain,and CD28, CD137, CD27 and CD3-zeta signaling domains, the inventionshould be construed to include any number of variations for each of thecomponents of the construct as described elsewhere herein. That is, theinvention includes the use of any antigen binding moiety in the CAR togenerate a CAR-mediated T-cell response specific to the antigen bindingmoiety. For example, the antigen binding moiety in the CAR of theinvention can target a tumor antigen for the purposes of treat cancer.

In some embodiments, the antigen bind moiety portion of the CAR of theinvention is designed to treat a particular cancer. For example, the CARmay be designed to target FRα with the signaling domains CD28, CD137,CD27 and CD3-zeta can be used to treat cancers and disorders includingbut are not limited to ovarian cancer, lung cancer, breast cancer, renalcancer, colorectal cancer, other solid cancers and the like. As notedabove, a CAR designed to target CD19 can be used to treat cancers suchas B-cell related cancer.

The CAR-modified T cells of the invention may also serve as a type ofvaccine for ex vivo immunization and/or in vivo therapy in a mammal.Preferably, the mammal is a human.

With respect to ex vivo immunization, at least one of the followingoccurs in vitro prior to administering the cell into a mammal: i)expansion of the cells, ii) introducing a nucleic acid encoding a CAR tothe cells, and/or iii) cryopreservation of the cells.

Ex vivo procedures are well known in the art and are discussed morefully below. Briefly, cells are isolated from a mammal (preferably ahuman) and genetically modified (i.e., transduced or transfected invitro) with a vector expressing a CAR disclosed herein. The CAR-modifiedcell can be administered to a mammalian recipient to provide atherapeutic benefit. The mammalian recipient may be a human and theCAR-modified cell can be autologous with respect to the recipient.Alternatively, the cells can be allogeneic, syngeneic or xenogeneic withrespect to the recipient.

The procedure for ex vivo expansion of hematopoietic stem and progenitorcells is described in U.S. Pat. No. 5,199,942, incorporated herein byreference, can be applied to the cells of the present invention. Othersuitable methods are known in the art, therefore the present inventionis not limited to any particular method of ex vivo expansion of thecells. Briefly, ex vivo culture and expansion of T cells comprises: (1)collecting CD34+ hematopoietic stem and progenitor cells from a mammalfrom peripheral blood harvest or bone marrow explants; and (2) expandingsuch cells ex vivo. In addition to the cellular growth factors describedin U.S. Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL-3 andc-kit ligand, can be used for culturing and expansion of the cells. Inaddition to using a cell-based vaccine in terms of ex vivo immunization,the present invention also provides compositions and methods for in vivoimmunization to elicit an immune response directed against an antigen ina patient.

Generally, the cells activated and expanded as described herein may beutilized in the treatment and prevention of diseases that arise inindividuals who are immunocompromised. In particular, the CAR-modified Tcells of the invention are used in the treatment of ovarian cancer. Incertain embodiments, the cells of the invention are used in thetreatment of patients at risk for developing ovarian cancer. Thus, thepresent invention provides methods for the treatment or prevention ofovarian cancer comprising administering to a subject in need thereof, atherapeutically effective amount of the CAR-modified T cells of theinvention.

The CAR-modified T cells of the present invention may be administeredeither alone, or as a pharmaceutical composition in combination withdiluents and/or with other components such as IL-2 or other cytokines orcell populations. Briefly, pharmaceutical compositions of the presentinvention may comprise a target cell population as described herein, incombination with one or more pharmaceutically or physiologicallyacceptable carriers, diluents or excipients. Such compositions maycomprise buffers such as neutral buffered saline, phosphate bufferedsaline and the like; carbohydrates such as glucose, mannose, sucrose ordextrans, mannitol; proteins; polypeptides or amino acids such asglycine; antioxidants; chelating agents such as EDTA or glutathione;adjuvants (e.g., aluminum hydroxide); and preservatives.

Compositions of the present invention are preferably formulated forintravenous administration.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

When “an immunologically effective amount”, “an anti-tumor effectiveamount”, “an tumor-inhibiting effective amount”, or “therapeutic amount”is indicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising the T cells described herein may be administered at a dosageof 10⁴ to 10⁹ cells/kg body weight, preferably 10⁵ to 10⁶ cells/kg bodyweight, including all integer values within those ranges. T cellcompositions may also be administered multiple times at these dosages.The cells can be administered by using infusion techniques that arecommonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng.J. of Med. 319: 1676, 1988). The optimal dosage and treatment regime fora particular patient can readily be determined by one skilled in the artof medicine by monitoring the patient for signs of disease and adjustingthe treatment accordingly.

In certain embodiments, it may be desired to administer activated Tcells to a subject and then subsequently redraw blood (or have anapheresis performed), activate T cells therefrom according to thepresent invention, and reinfuse the patient with these activated andexpanded T cells. This process can be carried out multiple times everyfew weeks. In certain embodiments, T cells can be activated from blooddraws of from 10 cc to 400 cc. In certain embodiments, T cells areactivated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc,80 cc, 90 cc, or 100 cc. Not to be bound by theory, using this multipleblood draw/multiple reinfusion protocol may serve to select out certainpopulations of T cells.

The administration of the subject compositions may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patientsubcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, by intravenous (i.v.) injection, orintraperitoneally. In one embodiment, the T cell compositions of thepresent invention are administered to a patient by intradermal orsubcutaneous injection. In another embodiment, the T cell compositionsof the present invention are preferably administered by i.v. injection.The compositions of T cells may be injected directly into a tumor, lymphnode, or site of infection.

In certain embodiments of the present invention, cells activated andexpanded using the methods described herein, or other methods known inthe art where T cells are expanded to therapeutic levels, areadministered to a patient in conjunction with (e.g., before,simultaneously or following) any number of relevant treatmentmodalities, including but not limited to treatment with agents such asantiviral therapy, cidofovir and interleukin-2, Cytarabine (also knownas ARA-C) or natalizumab treatment for MS patients or efalizumabtreatment for psoriasis patients or other treatments for PML patients.In further embodiments, the T cells of the invention may be used incombination with chemotherapy, radiation, immunosuppressive agents, suchas cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506,antibodies, or other immunoablative agents such as CAM PATH, anti-CD3antibodies or other antibody therapies, cytoxin, fludaribine,cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228,cytokines, and irradiation. These drugs inhibit either the calciumdependent phosphatase calcineurin (cyclosporine and FK506) or inhibitthe p70S6 kinase that is important for growth factor induced signaling(rapamycin). In a further embodiment, the cell compositions of thepresent invention are administered to a patient in conjunction with(e.g., before, simultaneously or following) bone marrow transplantation,T cell ablative therapy using either chemotherapy agents such as,fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, orantibodies such as OKT3 or CAMPATH. In another embodiment, the cellcompositions of the present invention are administered following B-cellablative therapy such as agents that react with CD20, e.g., Rituxan. Forexample, in one embodiment, subjects may undergo standard treatment withhigh dose chemotherapy followed by peripheral blood stem celltransplantation. In certain embodiments, following the transplant,subjects receive an infusion of the expanded immune cells of the presentinvention. In an additional embodiment, expanded cells are administeredbefore or following surgery.

The dosage of the above treatments to be administered to a patient willvary with the precise nature of the condition being treated and therecipient of the treatment. The scaling of dosages for humanadministration can be performed according to art-accepted practices. Thedose for CAMPATH, for example, will generally be in the range 1 to about100 mg for an adult patient, usually administered daily for a periodbetween 1 and 30 days. The preferred daily dose is 1 to 10 mg per dayalthough in some instances larger doses of up to 40 mg per day may beused (described in U.S. Pat. No. 6,120,766). Strategies for CAR T celldosing and scheduling have been discussed (Ertl et al, 2011, Cancer Res,71:3175-81; Junghans, 2010, Journal of Translational Medicine, 8:55).

EXPERIMENTAL EXAMPLES

Aspects of the invention are further described in detail by reference tothe following experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Example 1: CAR T Cell Engineering Targeting B Cell Malignancies

FIGS. 1-17 set forth a number of examples showing data that demonstratesa CAR embodiment can be used to treat and effectively cure B-cellmalignancies. The data is described further below.

The limitation of adoptive T cell therapy is MHC restriction. Tumorsescape by deletion of MHC-I and MHC-II to evade CD8 and CD4 T cells,respectively. Three keys to CAR-T therapy include (a) identifying targetantigen and obtaining scFv (b) high efficiency gene transfer into Tcells and (c) improved T cell signaling and expansion for efficacy andsafety.

It may be advantageous to use lentiviral vector to deliver CARs to Tcells for the following reasons:

-   -   a) Easy to prepare    -   b) Large payload, >9 kb    -   c) Integrate into dividing and non-dividing cells    -   d) Expanded tropism (VSV-G)    -   e) No viral genes expressed    -   f) No viral promoter (SIN—self inactivated)    -   g) No report of HIV-integration related cancer in        immunodeficiency (AIDS) patients

A CAR-4S lentivector was created (FIG. 1). The CAR-4S includedscFv-CD28-CD137-CD27-CD3z-2A-iCasp9. The scFV used was an anti-CD19scFV. The other domains were added to assist with co-signaling, T cellsurvival, T cell memory, effector activation and induced safety. The CARwas delivered to T cells and shown to target B-ALL cells (FIG. 2). A CART proliferation assay was performed and it was shown that the 4S CAR hada value of 33% at 6 days (FIG. 3). The anti-CD19 CAR T cells weredemonstrated to be capable of killing target CD19 leukemic cells, insome cases inducing apoptosis within 8 minutes (FIG. 4).

To improve the safety of CARs, a synthetic inducer of CAR dimerizationand apoptosis can be used, such as AP1903, which can interact with aFKBp-Casp9 (FIG. 5). Such “safety” CARs include 4S-CARs. Apoptosis canbe rapidly induced in 4S-CAR T cells (FIG. 6). These 4S-CARs may be usedin lenti-4S-CAR T cell therapy, such as by collecting blood andactivating T cells on day 1, performed CAR gene transfer on days 2-3,expanding the CAR T cells on day 4, and infusing CAR T cells into ahuman on day 5 to target a tumor in the human (FIG. 7). As aproof-of-concept, it was shown that lentiviral (LV) gene transfer intohuman T cells was efficient (FIG. 8). It was shown that these LV CAR Tcells expressed CD28 and CD8 or CD28 and CD27 on day 5 post genetransfer (FIG. 9).

Next, two case studies were performed on human patients. The details ofthe case study 1 patient are below:

-   -   a) 43 yr old Female—relapsed-refractory Ph+ ALL    -   b) 8-2007 diagnosed, hyper-CVAD: CR    -   c) 11-2007 allo-HSCT    -   d) BCR/ABL+: Gleevec    -   e) BCR/ABL>125%    -   f) ALL relapsed, imatinib resistant    -   g) Oral dasatinib, VDLD+IL-2/thymosin    -   h) Nilotinib resistant    -   i) Ponatinib trial, relapsed BCR/ABL    -   j) Relapsed (BM 35%)

The case study 1 patient received a CD19 CAR T infusion of 1.1×10⁸. TheCAR T infusion response at 7 days post infusion included a fever(39.7C), chest tightness, shortness of breath and coughing with bloodysputum. At 9 days post infusion, the patient had a fever (40C,non-infectious fever) and elevated LDH (1333 U/L vs. normal 114-240U/L). Plasma IgG was monitored post CAR-T infusion and IVIg wasadministered to the patient on day 19 after CAR infusion and every 3weeks thereafter (FIG. 10). Plasma IgA/IgM levels were also monitoredpost CAR-T infusion (FIG. 11). The case study continued with 3 totalinfusions: 0.6-3×10e7/kg. The patient was BCR/ABL negative at day 120.The levels of IL-6, interferon-gamma, CAR copy number in peripheralblood mononuclear cells (PBMCs), and CAR copy number in bone marrow (BM)was monitored (FIG. 12).

A second case study (case study 2) was performed. The details of thecase study 2 patient are provided below:

-   -   a) 34-year-old male    -   b) Presented with 2 wk history of abdominal mass    -   c) Biopsy: NHL, Burkitt's lymphoma        -   a. IHC: CD20(+++), PAX-5(+++), CD79a(+++), CD10(+),            Bcl-6(+),            -   Mum-1(−), Ki-67(+>75%)            -   b. FISH: Myc translocation

The treatment history of the case study 2 patient is shown in the tablebelow.

Regimen Response June to August R-CHOP × 3 PR September to DecemberHyper CVAD PD  (A + B) × 2 January   ICE × 1 PD February GEMOX × 1 PD

The CAR T cell infusion treatment protocol for the patient in case study2 included PBMC collection and CAR T cell preparation at day −7,fludarabine administration (25 mg/m2) on days −6 to −4, infusion of CART cells (6×10e7) on day 8, infusion of CAR T cells (6×10e7) on day 3,and infusion of CAR T cells (3.0×10e8) on day 7. The infusion responseon day 11 included:

-   -   a) Diaphoresis, anorexia, nausea, vomiting, hypoxia    -   b) Fever: 39.3° C.    -   c) Hypotension: BP 75/45 mmHg    -   d) HR: 150-180 bpm    -   e) Fluid resuscitation, infusion of dopamine

The serum uric acid and creatine levels of the patient were alsomonitored (FIG. 14), as were the level of serum cytokines IL-6 andinterferon-gamma (FIG. 15). The levels of IgG, IgA, and IgM were alsomonitored (FIG. 16). The clinical response of the patient was alsomonitored, and B cell reduction was observed (FIG. 17).

In summary, 4S generation CAR T cells targeted B-ALL with highefficiency, CAR T cells rapidly expanded and eradicated B cells in vivo,infused CAR T cells were capable of quickly reaching BM, and the 4S CARdesign was an important safety feature in patients.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

1-21. (canceled)
 22. A method for stimulating a T cell-mediated immuneresponse to a target cell population or tissue in a mammal, the methodcomprising administering to a mammal an effective amount of a cellgenetically modified to express a CAR, the CAR comprising an antigenbinding domain, a transmembrane domain, a costimulatory signalingregion, and a CD3 zeta signaling domain, wherein the costimulatorysignaling region comprises a CD28 signaling domain, a CD137 signalingdomain and a CD27 signaling domain; and wherein the antigen bindingdomain is selected to specifically recognize the target cell populationor tissue. 23-24. (canceled)
 25. The method of claim 22, wherein thegenetically modified cell is an autologous T cell.
 26. The method ofclaim 22, wherein the target cell population or tissue expresses a tumorantigen selected from the group consisting of CD19, CD20, CD22, ROR1,mesothelin, CD33/IL3Ra, c-Met, PSMA, Glycolipid F77, EGFRvIII, GD-2,NY-ESO-1 TCR, and MAGE A3 TCR.
 27. A method of treating a human withcancer, the method comprising administering to the human a T cellgenetically engineered to express a CAR, wherein the CAR comprises anantigen binding domain, a transmembrane domain, a costimulatorysignaling region, and a CD3 zeta signaling domain, wherein thecostimulatory signaling region comprises a CD28 signaling domain, aCD137 signaling domain and a CD27 signaling domain.
 28. The method ofclaim 27, wherein the human is resistant to at least onechemotherapeutic agent.
 29. A method of generating a persistingpopulation of genetically engineered T cells in a human diagnosed withcancer, the method comprising administering to the human a T cellgenetically engineered to express a CAR, wherein the CAR comprises anantigen binding domain, a transmembrane domain, a costimulatorysignaling region, and a CD3 zeta signaling domain, wherein thecostimulatory signaling region comprises a CD28 signaling domain, aCD137 signaling domain and a CD27 signaling domain; and wherein thepersisting population of genetically engineered T cells persists in thehuman for at least one month after administration.
 30. The method ofclaim 29, wherein the persisting population of genetically engineered Tcells comprises at least one cell selected from the group consisting ofa T cell that was administered to the human, a progeny of a T cell thatwas administered to the human, and a combination thereof.
 31. The methodof claim 29, wherein the persisting population of genetically engineeredT cells comprises a memory T cell.
 32. The method of claim 29, whereinthe persisting population of genetically engineered T cells persists inthe human for at least three months after administration.
 33. The methodof claim 29, wherein the persisting population of genetically engineeredT cells persists in the human for at least four months, five months, sixmonths, seven months, eight months, nine months, ten months, elevenmonths, twelve months, two years, or three years after administration.34-45. (canceled)